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selected relevant resistance genes for amplification detection in an additional 50 clinical isolates of carbapenem⁃resistant KPX,verified
their carriage status,and further elaborate on the sequencing results. Results:The drug sensitivity test showed that the KPX strain was
resistant to all antibiotics except colistin and tigecycline. The sequencing results relevant that the chromosomal gene sequence of this
bacterium is 5 468 925 bp in length and contains four plasmids(179 972 bp,141 377 bp,85 181 bp,and 20 247 bp). It had a total of 5
984 protein⁃coding genes,85 tRNA genes,and 25 rRNA operons. Additionally,this bacterium carried a large number of resistant genes
involved in encoding multiple antibiotics. MLST results indicated that the genetic type of this bacterium is ST11,and most of its
sequences are similar to previously reported strains in Sichuan and Hangzhou. The closest match was a strain with the code
SCKP020029(NCBI accession number:CP029384)of Klebislla. pneumoniae. The additional 50 carbapenem ⁃ resistant Klebislla.
pneumoniae collected were confirmed to produce extended⁃spectrum beta⁃lactamases(ESBLs),with 97% being of ST11. Pulsed⁃field
gel electrophoresis(PFGE)results showed that they belonged to three different clones. Among these strains,the detection rates of
resistance genes were as follows:KPC⁃2(98%),SHV⁃11(98%),TEM⁃1(76%),CTX⁃M(76%),Oqxb1(66%),qnrS(70%),Int1
(42%),sul1(82%),sul2(96%),iutA(88%),iucABCD(10%),and rmpA2(100%). Conclusion:The experimental results revealed
that a high degree of genomic similarity among nosocomial carbapenem resistant Klebislla. Pneumoniae strains,with epidemiological
characteristics of both clustering and sporadic cross⁃infection. Antimicrobial⁃resistance profiles suggested the presence of significant
selective antibiotic pressure. Furthermore,the dissemination of plasmid⁃mediated resistance genes among bacteria indicates that the
spread of these resistant strains within the hospital is primarily through plasmid transfer. Effective monitoring,isolation,and control of
these plasmids are essential measures to reduce the spread of bacterial resistance in nosocomial infections.
[Key words] carbapenem resistant;Klebislla. Pneumoniae;genome;sequencing
[J Nanjing Med Univ,2023,43(12):1729⁃1736,1763]
肺炎克雷伯菌(Klebsiella pneumonia,KPN)是临 (KPX),进行全基因组测序,根据测序结果在50株碳
床常见的细菌,常引起原发性肺炎、支气管炎、泌尿 青霉烯耐药的肺炎克雷伯菌中进一步进行基因测定
道感染。近年来,由于抗菌药物选择压力加大,临 和分型。
床滥用药物情况严重,致使多重耐药肺炎克雷伯菌
1 材料和方法
分离率明显增加。最近十年,肺炎克雷伯菌成为
“超级耐药”细菌并史无前例地迅速在全球传播,分 1.1 材料
[1]
离率仅次于大肠杆菌,排在第二位 。目前临床上 所有菌株均分离自临床标本,其中用于全基因
在常规药物无效的前提下都普遍认为碳青霉烯类 组测序的菌株 KPX 分离自 1 例 80 岁老年男性的痰
抗生素是治疗多重耐药肠杆菌科最有效的药物,但 液标本,该患者有长期住院史,患有严重的呼吸道
是随着碳青霉烯耐药肠杆菌(carbapenem⁃resistant 疾病。其余的 50 株碳青霉烯耐药的肺炎克雷伯菌
Enterobacterales,CRE)菌株的出现,尤其呈广泛耐药 株均来源于院内住院患者,年龄47~98岁,平均年龄
(extensively⁃drug resistant,XDR)或泛耐药(pandrug⁃ 78.5岁,男女比2.1∶1。所有试验菌株挑取单个菌落
resistant,PDR)特征的菌株检出率的逐年上升,导致 接种哥伦比亚琼脂 37 ℃培养过夜,长期保存则置
临床治疗陷入无药可用的困境。中国 CRE 监测网 于-80 ℃,使用前复苏。
数据显示 CRE 的粗死亡率为 33.5%,跟美国数据接 细菌的鉴定和药敏均使用法国梅里埃VITEK⁃2
近 。CRE几乎同时对所有抗生素耐药,因此,对产 COMPACT全自动系统,结果的判读参照CLSI⁃M100
[2]
碳青酶烯酶的多重耐药菌株进行研究显得尤为必要。 文件 。
[4]
泛耐药肺炎克雷伯菌是指对除粘菌素和替加 1.2 方法
环素外全部抗生素均耐药的菌株,这类菌株在临床 1.2.1 全基因组测序
上越来越多见,据报道产肺炎克雷伯菌碳青霉烯酶 对菌株 KPX 的全基因组 DNA 进行测序。首先
(Klebsiella pneumonia carbapenemase,KPC 酶)和新 使用SOAPdenovo v2.04预先组装Illumina测序数据,
德里酶(New Delhi metallo⁃β⁃lactamase,NDM 酶)是 然后使用blasR与Pacbio测序数据,使用序列之间的
碳青霉烯耐药的常见原因,但不局限于此 。因此 overlap 关系连接 scaffold,使用 CeleraAssembler 8.0
[3]
本实验中选择1株临床分离的泛耐药肺炎克雷伯菌 软件进行连接组装。 在所有 scaffold 连接完成后,