空肠弯曲菌脂寡糖核心区合成相关基因簇遗传多样性研究

目的分析我国空肠弯曲菌脂寡糖(LOS)核心区合成相关基因簇的序列特征,获得LOS核心区合成相关基因簇的特异DNA序列特征。方法选择132株空肠弯曲菌的全基因组序列,采用OrthoMCL软件,进行LOS核心区合成相关基因簇DNA的同源性分析并以特异型别基因簇的特异序列为靶位点,建立PCR方法,根据特异引物序列以及扩增产物大小,识别菌株LOS核心区合成相关基因簇的主要型别。结果在132株菌中,105株菌的LOS核心区合成相关基因簇的DNA序列属于14个已确定的LOS型别,27株菌为新型LOS,根据DNA的同源性可分为10个新型别,分别命名为CDC1~10。LOS合成相关基因序列中存在大量的多聚A/T、多聚G/C结构以及碱基的插入、缺失等突变,部分突变有可能导致LOS相关合成酶及转移酶的氨基酸序列发生改变,影响其抗原性。针对A、B、C、CDC8型LOS的特异DNA序列建立的PCR鉴定方法可在50株测序菌株的基因组中得到验证。结论10种LOS新型别的发现有利于更准确地鉴定空肠弯曲菌,解释LOS的抗原多样性特征。空肠弯曲菌的LOS核心区合成相关基因簇序列多样丰富,存在大量多聚结构和DNA序列的突变,是潜在的导致LOS抗原多样性的原因。主要LOS分型的PCR法可用于快速筛查5种特异LOS型,但仍需要更多菌株的验证。     

我国607株不同宿主来源弯曲菌耐药基因簇aadE-sat4-aphA-3分布分析

目的 了解不同宿主来源弯曲菌对氨基糖苷类抗生素的耐药现状,确定耐药菌株中氨基糖苷类耐药基因簇aadE-sat4-aphA-3的分布,并初步了解耐药菌株的菌型特征。方法 利用琼脂稀释法分析不同宿主来源菌株针对链霉素最小抑菌浓度(MIC),对耐药的221株菌通过聚合酶链反应筛查氨基糖苷类耐药基因簇aadE-sat4-aphA-3的分布情况。选取不同来源共100株耐药菌株进行多位点序列分型分析并进行聚类分析,获得耐药菌株的菌型特征及遗传相关性。结果 607株弯曲菌中共筛查到221株(36.41%)链霉素耐药株(MIC4 g/ml),其中腹泻患者来源菌株耐药率33.33%(78/234)、鸡来源菌株耐药率35.14%(123/350)、猪来源菌株耐药率86.96%(20/23)。空肠弯曲菌耐药率14.37%(51/355),结肠弯曲菌耐药率67.46%(170/252)。24株(10.86%)链霉素耐药弯曲菌中筛查到aadE-sat4-aphA-3耐药基因簇,分别来自腹泻患者14株、鸡4株、猪6株。100株耐药菌株共分为58种ST序列型。结论 结肠弯曲菌链霉素耐药率明显高于空肠弯曲菌,猪源弯曲菌耐药率显著高于人源和鸡源,空肠弯曲菌与结肠弯曲菌中均筛查到aadE-sat4-aphA-3耐药基因簇。     

深圳市南山区腹泻患者弯曲菌感染及病原学特征分析

目的获得本地区秋季腹泻患者弯曲菌的感染现状及病原学特征。方法收集深圳市南山区2018年9 — 11月腹泻患者的粪便标本,采用双孔滤膜法分离培养弯曲菌并应用荧光聚合酶链式反应（PCR）方法进行菌种鉴定。 应用脉冲场凝胶电泳（PFGE）和多位点序列分型（MLST）对分离菌株进行分子分型,应用琼脂稀释法获得菌株对11种抗生素的最小抑菌浓度（MIC）。 根据菌株的序列分型（ST）结果,结合既往菌株分型数据进行本地区菌株的遗传聚类分析。结果共采集腹泻患者粪便标本150份,其中男性患者74例,女性患者76例,年龄在1～86岁之间。 150份粪便标本中共分离到弯曲菌18株,检出率为12.00%（18/150）。 菌种鉴定发现,空肠弯曲菌占77.78%（14/18）,结肠弯曲菌占22.22%（4/18）。 14株空肠弯曲菌可分为11种PFGE带型,8个ST型别,以ST9763最多（35.71%,5/14）,是本研究新发现ST型别。 14株空肠弯曲菌对四环素全部耐药,对红霉素、阿奇霉素、泰利霉素、庆大霉素、链霉素及克林霉素100.00%敏感。 ST型别最小生成树聚类分析结果表明,不同宿主来源菌株没有显著聚集性,腹泻患者分离菌株分布在不同来源的组群中。结论采用双孔滤膜法提高了腹泻患者中弯曲菌的检出率。本地区弯曲菌的感染仍以空肠弯曲菌为主, 分离菌株对四环素类、喹诺酮类抗生素呈现较高的耐药特征,分离的空肠弯曲菌ST呈弥散分布特点。     

4种弯曲菌国家标准株实验室评价及应用分析

目的 依据《病原微生物菌(毒)种国家标准株评价技术标准》(WS/T 812—2022),对空肠弯曲菌、结肠弯曲菌、乌普萨拉弯曲菌和红嘴鸭(海鸥)弯曲菌4个菌种标准菌株病原特征进行实验室验证,评价应用标准菌株作为参比菌株进行弯曲菌检验标准实验室检测的可行性。方法 通过形态观察、生化试验及细菌动力学试验等方法获得标准菌株的菌落特点、形态特征、重要生化表型特征及运动特点;通过基因组测序获得菌株基因组水平遗传特征;通过琼脂扩散法和E-test法检测标准菌株的抗生素敏感性特征;通过空肠弯曲菌和结肠弯曲菌检验标准进行标准菌株作为参比菌种的应用评价。结果 标准菌株空肠弯曲菌NPRC(S) 01.13897、结肠弯曲菌NPRC(S) 01.13898、乌普萨拉弯曲菌NPRC(S) 01.13899和红嘴鸭弯曲菌NPRC(S)01.13900均符合弯曲菌对应菌种的病原特征,可以作为弯曲菌检验的参比菌株。结论 本研究中的标准菌株具有相应菌种的病原特征,可作为弯曲菌检测及菌种鉴定的参比菌株。     

多重PCR结合基因芯片技术检测11种致病菌方法的建立

目的建立一种运用多重PCR方法结合基因芯片技术快速、准确检测11种常见致病菌的方法。方法筛选志贺氏菌、肠炎沙门氏菌、伤寒沙门氏菌、大肠杆菌O157、副溶血性弧菌、普通变形杆菌、蜡样芽孢杆菌、金黄色葡萄球菌、单核细胞增生李斯特菌、产气荚膜梭菌、空肠弯曲菌的特异基因作为目的基因。设计相应的引物及探针,进行多重PCR扩增,制备寡核苷酸芯片。将多重PCR扩增产物与带有11种特异探针的基因芯片杂交。用扫描仪扫描,判定细菌种类。结果该基因芯片可特异性地检测11种致病菌,具有良好的特异性,基因组DNA检测灵敏度可达2×10-3ng。结论多重PCR结合基因芯片技术检测11种不同致病菌的方法特异性好,灵敏度高,具有较好的实用性。     

斯氏弓形杆菌基于基因组序列遗传特征分析

  目的  获得斯氏弓形杆菌的遗传特征,并对其耐药基因及毒力相关基因进行预测分析。  方法  对本实验室分离培养的8株斯氏弓形杆菌进行基因组测序并从NCBI和PATRIC数据库中下载全部已完成基因组测序的15株斯氏弓形杆菌的全基因组序列,应用生物信息学软件对23株不同地域、不同宿主来源的斯氏弓形杆菌进行遗传特征分析。  结果  23株斯氏弓形杆菌中仅3株菌携带耐药基因,且均为β-内酰胺类抗生素耐药基因。 所有菌株均含有与免疫逃避、侵袭及运动等相关的毒力基因。 与其他菌种弓形杆菌常见的10种毒力基因比对分析发现,本研究中所有斯氏弓形杆菌菌株均含有ciaB、tlyA、cj1349、pldA和mviN基因,2株菌（CNAS12-1和F28）含有iroE基因,均不携带另外4种毒力基因。 21.7%（5/23）的菌株包含Ⅵ型分泌系统（T6SS）基因簇,且其与弯曲菌属内的T6SS存在基因组成及排列的差异。  结论  斯氏弓形杆菌有较高的遗传多样性,部分菌株可能有潜在的耐药和致病特征。     

不同方法检测食源性致病菌的对比研究

目的探讨基因芯片技术和多重PCR技术用于检测和筛查食源性致病菌的可行性。方法通过设计靶细胞引物序列,采用生物素标记反向引物5′端,氨基基团标记寡核苷酸探针5′端。将探针在固相载体上点样,制备基因芯片,PCR产物与芯片点制探针区域进行杂交,并对PCR杂交反应的体系进行优化。结果基因芯片技术可以同时检测志贺氏菌、沙门氏菌、肺炎克雷伯菌、布鲁氏菌、奇异变形菌、金黄色葡萄球菌和空肠弯曲菌等多种病原菌,操作简便,特异性强。细菌纯培养物灵敏度为5.0×102 CFU/mL,DNA检测灵敏度为0.1pg,检测分离菌株符合率为100%。利用引物建立和优化了PCR检测体系,分别确定了Mg2+浓度和退火温度Tm值为1.5mmol/L和56℃,检测灵敏度达到10pg,此灵敏度下可以扩增出全部特异性引物条带。结论通过基因芯片技术和多重PCR技术可以有效检测食源性致病菌,为高通量筛查检测病原菌提供了新思路,值得在食品安全领域推广应用。     

碳青霉烯耐药肺炎克雷伯菌ST11菌株的基因组学特点分析

目的分析碳青霉烯耐药肺炎克雷伯菌(CRKP)ST11菌株的基因组学特点。方法挑取南京地区流行的3株CRKP-ST11菌株,提取基因组DNA,进行全基因组学测序,获得拼接后的全基因组。同时,从NCBI数据库中下载HS11286(中国上海)、JM45(中国杭州)、ATCC BAA-2146(美国/印度)的基因组为对照菌株,进行基因组GC碱基含量、单核苷酸多态性、亲缘关系及荚膜多糖(CPS)基因簇结构分析。结果CPS基因簇的G+C含量大多<50%,不同于染色体其他部分,是CRKP-ST11的主要单核苷酸多态性位点;进化树分析显示南京地区分离的3株CRKP-ST11菌株与上海、杭州的CRKP-ST11亲缘关系相近,与美国/印度的CRKP-ST11较远。这3株CRKP-ST11的CPS基因簇一致,与杭州、上海及美国/印度的3株CRKP-ST11菌株的差异主要在CPS可变区。结论CPS是CRKP-ST11的主要单核苷酸多态性位点,可能是CRKP-ST11菌株的主要进化区,CRKP-ST11的流行可能与CPS基因簇可变区有关,具有地域特点。     

应用TaqMan实时荧光-聚合酶链反应方法快速检测粪便标本空肠弯曲菌的研究

目的建立空肠弯曲菌TaqMan实时荧光-PCR方法,用于粪便标本的直接检测。方法根据空肠弯曲菌特异性基因hipO和mapA分别设计引物和探针,在对2组引物和探针进行灵敏度、特异性和重复性评价的基础上,对45例临床腹泻患者粪便标本提取DNA之后,荧光PCR检测,同时进行分离培养。 结果两组引物和探针能准确检测空肠弯曲菌菌株2株,检测限可达到10~20 cfu/ml,并与其他肠道致病菌无交叉反应。检测45份腹泻病例粪便标本,该方法检测到3份为阳性,同时进行的传统培养方法仅从该3份标本中的两份中分离到空肠弯曲菌。 结论本研究建立的TaqMan荧光PCR检测粪便标本中所携带的空肠弯曲菌灵敏度高,特异性好,能够提高粪便中空肠弯曲菌的阳性检出率和缩短检测时限。     

多重耐药肺炎链球菌差异DNA消减文库的构建及初步筛选

目的比较肺炎链球菌多重耐药株与敏感株标准参考菌之间的差异基因。方法使用抑制性消减杂交技术构建多重耐药肺炎链球菌差异DNA消减文库,经斑点杂交筛选阳性克隆后对部分DNA片段进行测序和同源分析。结果成功的构建了肺炎链球菌多重耐药株差异DNA消减文库,经斑点杂交的初步筛选,有17个仅能与多重耐药株DNA探针杂交,而不能与敏感株DNA探针杂交。对这17个克隆片段测序及基因库检索分析,发现2个未知新序列。结论从全基因角度研究肺炎链球菌多耐药菌株与标准菌株基因组DNA分子遗传差异,为发现、克隆肺炎链球菌多重耐药相关基因提供依据。     

DNA probes for identification of tetracycline resistance genes in Campylobacter species isolated from swine and cattle

Tetracycline-resistant strains of Campylobacter jejuni and Campylobacter coli from swine and cattle colons were isolated and characterized by hybridization with DNA probes. A probe consisting of the 1.8-kilobase (kb) HincII fragment from pUA466 was highly specific for the detection of tetracycline resistance (Tcr) in C. jejuni and C. coli. The 5-kb tetM DNA probe from Streptococcus agalactiae plasmid pJI3 which has homology with the 1.8-kb HincII fragment from pUA466 could also be used to detect Tcr Campylobacter strains. However, the tetM probe had a much lower sensitivity and required a lower stringency of hybridization. Therefore, the 1.8-kb HincII fragment appeared to be more appropriate for the classification of Tcr in Campylobacter spp. No homology was detected between the Tcr determinant from Campylobacter spp. and the tetL and tetN probes from Streptococcus spp. DNA homology was demonstrated between pUA649, a derivative of plasmid pUA466 which had lost most of the Tcr region, and Tcr plasmids from C. jejuni and C. coli isolated from animal and human sources. There was also homology between pUA649 and the chromosomes of C. jejuni and C. coli strains. In this study, all but one of the tetracycline-resistant C. coli and C. jejuni strains contained plasmids of approximately 50 kb which hybridized with the 1.8-kb HincII probe. In one C. coli strain (UA703), Tcr appeared to be chromosomally mediated.     

Molecular cloning of a species-specific DNA probe for Campylobacter jejuni

Conventional procedures for isolating and identifying Campylobacter jejuni are cumbersome and time-consuming. A simpler approach would be to use DNA probes to identify these organisms. To obtain such probes we cloned chromosomal DNA from C. jejuni into the lambda replacement vector EMBL 4. Recombinant phages were screened for C. jejuni-specific inserts by DNA hybridization using chromosomal DNA from either C. jejuni or C. coli which had been radioactively labelled with 32P. By this means, recombinant phages were identified which hybridized to C. jejuni but not to C. coli DNA. These phages were then subjected to further screening using DNA from other Campylobacter species. Three DNA fragments were identified which hybridized to DNA from eight ATCC (American Type Culture Collection) strains of C. jejuni but not to DNA from C. coli, C. laridis, C. fetus or a variety of other bacterial species. These DNA fragments are suitable for use as specific probes in DNA-based diagnostic tests for C. jejuni infections.     

Campylobacter genes responsible for the development and determinant of clinical features of Guillain-Barré syndrome

Progress has been made in Guillain-Barré syndrome, a post-infectious autoimmune neuropathy, especially on identifying Campylobacter jejuni genes responsible for the development and determinant of clinical features. C. jejuni strains carrying a sialyltransferase gene (cst-II), which is essential for the biosynthesis of ganglioside-like lipooligosaccharides, are associated with the development of Guillain-Barré syndrome. The C. jejuni sialyltransferase (Cst-II) consists of 291 amino acids, and the 51st determines its enzymatic activity. Strains with cst-II (Thr51) expressed GM1-like and GD1a-like lipooligosaccharides, whereas strains with cst-II (Asn51) expressed GT1a-like and GD1c-like lipo-oligosaccharides. Patients infected with the cst-II (Thr51) strains had anti-GM1 or anti-GD1a IgG antibodies, and showed limb weakness. Patients infected with the cst-II (Asn51) strains had anti-GQ1b IgG antibodies, and showed ophthalmoplegia and ataxia. The cst-II gene is responsible for the development of Guillain-Barré and Fisher syndromes, and the polymorphism (Thr51/Asn51) determines which syndrome develops after C. jejuni enteritis.     

Detection of two different kanamycin resistance genes in naturally occurring isolates of Campylobacter jejuni and Campylobacter coli.

A total of 225 isolates of Campylobacter jejuni and 54 isolates of Campylobacter coli were screened for resistance to kanamycin. Among these, five resistant isolates of C. jejuni and six resistant isolates of C. coli, all with different plasmid patterns, were identified. Each contained at least one plasmid greater than or equal to 41 kilobases in size. The MIC of kanamycin for all 11 strains was determined to be greater than or equal to 256 micrograms/ml by an agar dilution method. In addition, all of the strains exhibited resistance to tetracycline (greater than or equal to 16 micrograms/ml). Eight of the 11 strains transferred the kanamycin resistance phenotype to other Campylobacter strains by conjugation. DNA from 9 of the 11 strains hybridized to a DNA probe specific for the 3'-O-aminoglycoside phosphotransferase type III gene. The remaining two strains also failed to show homology with DNA probes specific for the genes encoding 3'-O-aminoglycoside phosphotransferase types I, II, and III. The novel kanamycin resistance gene was cloned into the vector pBR322 and was expressed in Escherichia coli. Phosphocellulose paper binding assays on sonicates of the E. coli strain carrying the cloned kanamycin determinant demonstrated significant activity against kanamycin, neomycin, and amikacin but not against butirosin, gentamicin, tobramycin, or lividomycin, suggesting that the enzyme is the product of a 3'-O-aminoglycoside phosphotransferase type of aminoglycoside resistance gene.     

Tetracycline resistance of Australian Campylobacter jejuni and Campylobacter coli isolates

OBJECTIVES: Tetracycline resistance in Campylobacter is encoded by the tet(O) gene and is usually associated with conjugative plasmids. Little was known about tetracycline resistance in Australian Campylobacter species, therefore we investigated this resistance in 41 Campylobacter jejuni and five Campylobacter coli strains from humans and healthy chickens. METHODS: Tetracycline MICs were determined for each isolate using an agar dilution method. The distribution and localization of tet(O) on plasmid and chromosomal DNA was determined by Southern-blot experiments. The ability to transfer resistance to recipient strains was examined through conjugation studies. Identity of transconjugants was confirmed by PCR and flaA-restriction fragment length polymorphism analysis. RESULTS: High-level tetracycline resistance was observed, ranging from 32 to >256 mg/L. Plasmids were detected in 74% of isolates with plasmids between 30 and 40 kb in size most frequently isolated. tet(O) was present in all tetracycline-resistant isolates. In the majority of strains under study the tet(O) gene was chromosomally encoded. Tetracycline resistance of six C. jejuni strains in which tet(O) was plasmid mediated was transferred by conjugation to a C. jejuni recipient strain. Transfer did not occur between tetracycline-resistant C. jejuni strains and a C. coli recipient. No difference in MICs, plasmid carriage and tet(O) localization was detected between human and chicken isolates. CONCLUSIONS: These data indicate that the tet(O) gene, previously reported in Campylobacter strains throughout the world, is present in Australian Campylobacter. This study will lead to a greater understanding of antibiotic resistance distribution in Campylobacter spp. in Australia.     

Classification of Campylobacter strains using DNA probes

DNA homology determinations were used to classify atypical strains of Campylobacter spp. and Campylobacter-like organisms. Using the slot-blot hybridization technique, atypical strains were classified as C. coli or C. jejuni. Some of the Campylobacter-like organisms isolated from animals were catalase-positive, but they did not hybridize with the DNA probes prepared from the total genomic DNA of C. coli, C. jejuni, C. laridis, C. fetus subsp. fetus and C. hyointestinalis. The taxonomic classification of these strains remains uncertain. The slot-blot hybridization technique was simplified by applying whole cells from a 24 h broth culture, instead of pure DNA to nitrocellulose membranes. The results of DNA homology from both methods were comparable, and the classification of these organisms using these methods was the same. Therefore, the dot-blot hybridization method is recommended as a more economical and time-saving method for use in the taxonomic classification of atypical Campylobacter spp.     

Immunoblot and enzyme-linked immunosorbent assays of Campylobacter major outer-membrane protein and application to the differentiation of Campylobacter species

The major outer-membrane protein (MOMP) from Campylobacter jejuni NCTC 11168 was purified by solubilization in Triton X-100. Whole-cell proteins of Campylobacter species and the MOMP were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, immunoblotting on nitrocellulose paper and enzyme-linked immunoblot assay (ELISA) analyses using polyclonal antiserum to the C. jejuni MOMP. Purified MOMP from C. jejuni NCTC 11168 contained a single major protein of 46 kDa. Whole-cell preparation of C. jejuni NCTC 11168 also showed a major band of 46 kDa which was recognized in immunoblots by the anti-MOMP serum. Other C. jejuni strains, as well as C. coli and C. laridis strains showed similar MOMP bands at 45-46 kDa. Other Campylobacter species (C. fetus ss. fetus, C. hyointestinalis and C. pylori (new nomenclature] did not react in immunoblots with antiserum to the C. jejuni MOMP. ELISAs showed that antiserum raised against the C. jejuni MOMP reacted with C. jejuni, C. coli and C. laridis strains. Other Campylobacter species displayed only a very low degree of cross-reactivity. The distinct antigenic relationship demonstrated between the MOMP of C. jejuni, C. coli and C. laridis is consistent with the close degree of relatedness of these species as determined by DNA homology studies. The anti-MOMP serum appeared to be useful in the rapid differentiation of C. jejuni, C. coli and C. laridis from other Campylobacter species.     

PCR identification of Campylobacter coli and Campylobacter jejuni by partial sequencing of virulence genes

The objective of this study was to utilize a multiplex PCR assay for concurrent detection of Campylobacter spp. and C. coli or C. jejuni, using probes derived from genes cadF and ceuE and an undefined virulence gene. A total of 97 Campylobacter strains, isolated from turkey litter (n=74), chicken livers (n=15) and clinical (n=8) samples, were speciated using the PCR-based assay. PCR amplification of the isolates identified a 400-bp cadF gene, conserved in Campylobacter species, an 894-bp ceuE gene, specific for C. coli, and a 160-bp oxidoreductase gene, specific for C. jejuni. The approximately 35 kDa cadF adhesion proteins allow Campylobacter to bind to the intestinal epithelial cells and the 37 kDa ceuE lipoproteins are involved in siderophore transport. Sequencing of the 160-bp undefined gene yielded a 67% protein identical match with a gene encoding an oxidoreductase subunit in C. jejuni. The specificity of the assay was validated on 36 non-Campylobacter strains (11 Gram-positive and 25 Gram-negative bacteria). The PCR assay identified 59% of turkey and 47% of chicken isolates as C. jejuni, and 41% of turkey and 53% of chicken isolates as C. coli. All human isolates were identified as C. jejuni. The specificity of this assay to detect C. coli or C. jejuni was 97%.     

Antimicrobial resistance of Campylobacter jejuni and Campylobacter coli with special reference to plasmid profiles of Japanese clinical isolates.

A total of 111 clinical isolates of Campylobacter jejuni and 10 clinical isolates of Campylobacter coli were characterized by their susceptibility to nine antimicrobial agents and by their plasmid profiles on agarose gel electrophoresis. All of the C. jejuni isolates were susceptible to chloramphenicol, ciprofloxacin, erythromycin, kanamycin, and nalidixic acid, but 55% were tetracycline resistant. In the 10 C. coli isolates, a high prevalence of multiple-antibiotic resistance was noted. Plasmids were found in 82% of the tetracycline-resistant and 15% of the tetracycline-susceptible C. jejuni isolates. Tetracycline resistance in six randomly selected C. jejuni isolates, which contained 50- or 135-kilobase (kb) plasmids, was transferred by conjugation to a Campylobacter fetus subsp. fetus recipient with recovery of a 50- or a 45-kb plasmid from transconjugants. From one multiple-antibiotic-resistant C. coli isolate, resistance to tetracycline, kanamycin, and chloramphenicol was transferred concomitantly with a 58-kb plasmid, pNR9589. Nonconjugative 98-kb plasmids, pNR9131 and pNR9581, from C. coli isolates with resistance to tetracycline, kanamycin, and erythromycin were shown by cloning experiments to code for at least kanamycin resistance. Restriction digests revealed that 50-kb plasmids from tetracycline-resistant C. jejuni isolates were identical, although plasmids from multiple-antibiotic-resistant C. coli isolates shared partial DNA homology to each other. Cloning of the kanamycin and chloramphenicol resistance genes of pNR9589 into Escherichia coli showed that the two genes are closely linked or clustered. Double-digestion analysis of the fragments encoding the kanamycin resistance of pNR9131, pNR9581, and pNR9589 showed that these three plasmids contain a common fragment related to kanamycin resistance.