A physical and genetic map of the IncFI plasmid ColV2-K94

A restriction enzyme map of the IncFI plasmid ColV2-K94 was generated using EcoRI, BamHI, HindIII, and XhoI; the genetic features of this element were then mapped from previous heteroduplex studies.     

Isolation of a conjugative plasmid in Escherichia coli determining formaldehyde resistance

A clinical isolate of Escherichia coli which was resistant to the disinfectant formaldehyde was investigated. The strain harboured a plasmid of 62 MDa size. It was shown by conjugation, transformation and plasmid-curing experiments that the formaldehyde resistance is plasmid-mediated and transferable to other strains.     

Characterization of the stability functions and inverted repeat structure X3 of the IncFI plasmid ColV2-K94

A region of the IncFI plasmid ColV2-K94 which showed homology to the sop partitioning genes of F was cloned and characterized in an attempt to study the stability functions of this element. The sop region contained the incD incompatibility determinant common to many IncFI plasmids, but could not confer on ColV2-K94 miniplasmids the same stable inheritance found in the intact ColV2-K94; thus, other functions appear to be required for efficient plasmid maintenance. Adjacent to the area of sop homology was the X3 region, which was found to contain three inverted IS1-like sequences. The X3 region of ColV2-K94 was similar in organization to the aerobactin iron uptake region of ColV3-K30, but ColV2-K94 lacked the ability to synthesize either the aerobactin siderophore or its outer membrane receptor.     

Transport of nucleic acid bases and nucleosides across the bacterial membrane

Abstract Defects have been found in a colorimetric assay developed by its inventors for determining rapidly and quantitatively whether strains of Escherichia coli are resistant to the lethality of mammalian serum. Results gained with the colorimetric assay on the serum resistance of E. coli strains bearing colicin V plasmids differed from results gained with an assay in which the rate of growth of the bacterial strains in a medium containing rabbit serum was estimated turbidimetrically. The colorimetric assay was intended by its developers to facilitate studies of the genetics of serum resistance in pathogenic bacteria. When used as originally described, the assay is likely to mislead in such studies.     

The role of capsular antigens in serum resistance and in vivo virulence of Escherichia coli

Abstract To identify critical microbial determinant(s) in serum resistance and in vivo virulence, 5 strains of Escherichia coli with different combinations of K and O types were examined along with their unencapsulated mutants. All 3 E. coli strains possessing the K1 capsule were considerably more resistant to normal human serum in vitro and more virulent in newborn rats than their isogenic mutants lacking the K1 capsule. In contrast, two K5 encapsulated strains and their unencapsulated mutants were essentially the same for their degree of serum resistance and in vivo virulence. These findings suggest that for the demonstration of serum resistance and in vivo virulence, a capsule is required for E. coli strains encapsulated with K1, whereas certain E. coli strains encapsulated with K5 may not need a capsule.     

Virulence genes,antibiotic resistance and plasmid profiles of Enterococcus faecalis and Enterococcus faecium from naturally fermented Turkish foods

Aim: To determine the virulence genes, antibiotic resistance and plasmid profiles of 16 Enterococcus faecium and 68 Enterococcus faecalis strains isolated from various naturally fermented foods. Methods and Results: The presence of virulence genes (agg₂, gelE, cylM, cylB, cylA, espfs, espfm, efaAfs, efaAfm, cpd, cop, ccf, cad) and also the genes vanA and vanB were investigated by polymerase chain reaction (PCR). Antibiotic resistance of the isolates was determined by disc diffusion method. Most of the tested isolates were positive for virulence genes and resistant to some antibiotics. One of the Ent. faecalis strains isolated from a cheese sample carried the vanA gene and was intermediately resistant to vancomycin. The strains usually contained large plasmids, which might harbour acquired antibiotic resistance. Conclusion: The study showed that Ent. faecium and Ent. faecalis strains isolated from naturally fermented Turkish foods may be potential risk factors for consumer health in terms of virulence genes and acquired antibiotic resistance. Significance and Impact of the Study: The results indicate the importance of enterococcal contamination in terms of the safety of some fermented Turkish foods.     

IncFII-FIA-FIB型多重耐药质粒pBTR-CTXM的结构基因组学分析

【背景】IncFII-FIA-FIB型质粒广泛存在于肠杆菌科细菌中,介导了许多耐药基因的水平转移,并导致细菌多重耐药问题日益严重。【目的】分析IncFII-FIA-FIB型多重耐药质粒pBTR-CTXM的基因组结构,并研究其介导大肠杆菌BTR株的耐药基因水平转移机制。【方法】利用PCR进行耐药基因筛查;接合转移和电转化实验验证质粒pBTR-CTXM是否具备自主接合转移的特性;VITEK 2 Compact全自动细菌鉴定及药敏分析仪测定相关菌株对抗生素的药物敏感性;构建MatePair文库并进行细菌全基因组高通量测序和质粒结构基因组学分析。【结果】菌株BTR是携带blaNDM-1、blaCTX-M-15、blaTEM、qnrD、qnrS1、mph(A)、erm(B)和tetA(B)等耐药基因的多重耐药大肠杆菌,其中blaCTX-M-15、mph(A)、erm(B)和tet A(B)等耐药基因均位于大小为144 939 bp的质粒p BTR-CTXM (GenBank登录号MF156697)上,该质粒可与菌株BTR内质粒pNDM-BTR接合共转移到受体菌大肠杆菌EC600中。pBTR-CTXM具备IncFII-FIA-FIB型质粒典型的骨架区结构,其多重耐药(Multidrug-resistant,MDR)区由新的复合型转座子Tn6492、Tn2残余、Tn10残余、ISEcp1-blaCTX-M-15-Δorf477转座单元和一些插入序列组成。【结论】pBTR-CTXM中新复合型转座子Tn6492与Tn10残余和ISEcp1-blaCTX-M-15-Δorf477转座单元共同介导大肠杆菌BTR株的多重耐药与耐药基因的水平传播。     

The contribution of tellurite resistance genes to the fitness of Escherichia coli uropathogenic strains

The presence of tellurite resistance gene operons has been reported in several human pathogens despite the fact that tellurium, as well as its soluble salts, are both rare in nature and are no longer in use as antimicrobial agents. We have introduced the cloned terWZA-F genes from an uropathogenic Escherichia coli isolate into another clinical E. coli isolate that was shown to be ter-gene free. The presence of the introduced genes increased the level of potassium tellurite resistance, as well as the level of resistance to oxidative stress mediated by hydrogen peroxide; and prolonged the ability of particular strains to survive in macrophages. We therefore propose that the contribution of tellurite resistance genes to oxidative stress resistance in bacteria is at least one reason for their presence in the genomes of a broad range of pathogenic microorganisms.     

Prevalence and antibiotic resistance of <Emphasis Type="Italic">Escherichia coli</Emphasis> in tropical seafood

Summary The occurrence and antibiotic resistance of Escherichia coli in tropical seafood was studied. A 3-tube MPN method was used for determining the level of faecal contamination of fresh and processed seafood. Of the 188 samples tested which included finfish, shellfish, water and ice, 155 were positive for the presence of faecal coliforms following incubation at 44.5 °C. However, E. coli was isolated from only 47% of the samples positive for faecal coliforms. The antibiotic resistance of 116 strains isolated from seafood was tested using 14 different antibiotics including ampicillin, cephalothin, chloramphenicol, ciprofloxacin, gentamycin, nalidixic acid, streptomycin and vancomycin. Seven strains were resistant to more than five antibiotics of which one was resistant to eight antibiotics. The multiple drug resistant strains harboured plasmids of varying sizes. Antibiotic susceptibility studies revealed that seafood from India contains multiple antibiotic resistant strains of E. coli which may serve as a reservoir for antibiotic resistance genes in the aquatic environment. All the strains used in this study did not harbour any virulence genes commonly associated with pathogenic E. coli, when tested by polymerase chain reaction (PCR).     

急性腹泻患者致泻性大肠埃希菌流行情况及耐药性分析

目的 了解致泻性大肠埃希菌（DEC）在急性腹泻患者中的分布及对常用抗菌药物的敏感性,为疾病的防控和治疗提供依据。方法 收集2014年1月至2015年12月期间就诊于浙江大学医学院附属第一医院急性腹泻患者粪便标本,采用常规病原菌检验流程对常见肠道致病菌进行分离培养鉴定,分离到的疑似大肠埃希菌采用多重PCR和单重PCR进行鉴定、分型,采用纸片扩散法（K-B法）对各型DEC进行18种常用药物敏感试验。结果 从1 019例急性腹泻患者标本中分离到396株病原菌,其中DEC 230株,分离率为22.6%,占所有分离菌株的58.1%,居病原菌首位。230株DEC中,ETEC占56.5%,EAEC占30.4%,EPEC占8.7%,STEC占0.4%,混合型DEC占0.9%,未发现EIEC。各型DEC全年均有检出,其中夏季（6~8月）分离率最高,达24.0%,患者主要分布在19~45岁,男女检出率分别为21.8%和23.2%,差异无统计学意义。DEC对氨苄西林的耐药率最高,达49.3%,对头孢唑林、复方新诺明、氨苄西林/舒巴坦的耐药率分别为46.4%、35.7%、30.9%,其余抗菌药物耐药率均低于15.0%,未出现亚胺培南耐药株。207株DEC中,产ESBLs菌株占41.1%,多重耐药菌株占44.9%。结论 DEC是本地区急性腹泻最常见的细菌性病原,是临床肠道感染中不可忽视的病原之一,尤其是同时携带多型别毒力基因菌株及产ESBLs菌株和多重耐药菌。长期持续监测其分布及耐药趋势,对疾病的预防、治疗具有重要意义。     

Variation and modeling of the probability of plasmid loss as a function of growth rate of plasmid-bearing cells of Escherichia coli during continuous cultures

A large number of models concerning cultures of genetically engineered bacteria have been described. Among them, some are specifically adapted to continuous cultures and lead to the determination of two variables: (i) the difference in the specific growth rates between plasmid-carrying cell and plasmid-free cells (deltamu) and (ii) the frequency of plasmid loss by plasmid-containing cells (p(r)mu(+)). Until now, studies have been performed on the global expression p(r)mu(+) and deltamu, whose value during continuous assays have been supposed approximately constant (mean value) and not on separate values of both terms p(r) and mu(+), respectively, probability of plasmid loss and specific growth rate of the plasmid-carrying cells. So far these studies do not allow examination of the relationship between these two last parameters. Experimental results were obtained with Escherichia coli C600 galk (GAPDH), a genetically engineered strain that synthetizes an elevated quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). From data obtained during continuous cultures, it is shown that during an assay, deltamu, and p(r)mu(+) do not remain constant. An appropriate mathematical analysis of the expression of mu(-) (specific growth rate of the plasmid-free cells) and mu(+) has been built up. This allows the evaluation of the values of mu(+) and mu(-) during the continuous cultures carried out at different dilution rates. Values of p(r) have been calculated from these data. Indeed our results show that p(r) increases with mu(+). A modeling approach which allows correct simulation of this variation is also proposed. This model is derived from the Hill equation regarding cooperative binding of enzymic type reaction. (c) 1993 John Wiley & Sons, Inc.     

Whole Genomic analysis of a clinical isolate of Uropathogenic Escherichia coli strain of Sequence Type - 101 carrying the drug resistance NDM-7 in IncX3 plasmid

The emerging NDM-producing Enterobactereciae is a major threat to public health. The association of NDM-7 with sequence type 101 E.coli is identified in very few numbers. Therefore, it is of interest to analyse the whole genome sequence of NDM-producing uropathogenic E. coli XA31 that was found to carry numerous drug resistance genes of different antibiotic classes. The isolate E. coli belongs to ST-101 carrying blaNDM-7 coexisting with several resistance genes blaOXA-1, blaTEM1-A, blaCTX-M15, aac(6'')-Ib-cr, catB3, tetB. Resfinder predicts this and four other plasmid replicons were identified using the Plasfinder in the CGE platform. The high transferable IncX3 plasmid was found to carry the NDM-7 gene. Thus, we the report the combination of NDM-7-ST101-IncX3 in India. The combination of this epidemic clone with NDM-7 is highly required to develop an effective infection control strategy.     

Construction of new shuttle plasmid vectors for Escherichia coli-Bacteroides transgeneric cloning

Abstract An Escherichia coli-Bacteroides shuttle vehicle (pKBF367-1) was constructed by combining the pBR322 derivative pKC7 (5.9 kb) with [1] a 4.6 kb cryptic plasmid from Bacteroides fragilis ; and [2] the 4.2 kb Eco RI-B fragment of the B. fragilis plasmid pBFTM10. This latter component allowed selection of clindamycin-resistant transconjugants upon helper plasmid-mediated transfer to a recipient strain of Bacteroides distasonis . To improve the potential of pKBF367-1 (14.7 kb) as cloning vector, successive deletions generated derivatives of 12.8, 10.5 and 9.3 kb, which were still able to replicate in B. distasonis 419. These bifunctional vectors were successfully employed to introduce transposon Tn 501 (Hg^r) into B. distasonis 419, but expression of mercury resistance was not observed. This plasmid vehicles series may be useful for cloning Bacteroides genes in E. coli and studying their expression in a heterologous Bacteroides strain.     

大肠埃希菌连续分离株氨基糖苷类修饰酶基因研究

目的了解临床分离的大肠埃希菌耐药性及氨基糖苷类修饰酶（AMEs）基因的存在状况。方法测定临床分离的60株大肠埃希菌对19种抗菌药物的敏感性,采用PCR技术检测氨基糖苷类修饰酶基因。结果60株大肠埃希菌呈现多重耐药,氨基糖苷类修饰酶基因aac（3）-Ⅱ、aac（6′）-Ⅰb、aac（6′）-Ⅱ、ant（3′′）-Ⅰ、ant（2′′）-Ⅰ的阳性率分别为36.7%、18.3%、0%、10%、1.6%。携带1种或1种以上基因的菌株有33株（55%）。结论临床分离的大肠埃希菌多重耐药严重,氨基糖苷类修饰酶基因携带率较高。     

Increased stability of maintenance of pAT153 in Escherichia coli HB101 due to transposition of IS1 from the chromosome into the tetracycline resistance region of pAT153

Abstract The plasmid vector pAT153 was rapidly lost from carbon-limited continuous cultures of Escherichia coli HB101 (pAT153) at a dilution rate of 0.15 h⁻¹. In one experiment, the plasmid was maintained by 80% of the host bacteria for up to 35 generations. The tetracycline-resistance gene was not expressed from the majority of the plasmid DNA in this population of E. coli HB101 due to transposition of IS1 from the bacterial chromosome into the aminoterminal region of the tet gene of pAT153. This plasmid, pLCX1, when isolated and retransformed into E. coli HB101, was more stably maintained than pAT153. Similar plasmids have been isolated from other glucose, phosphate, ammonium and sulphate-limited chemostats.     

2009—2017年大肠埃希菌临床分布及耐药性分析

目的了解大肠埃希菌(Escherichia coli,E.coli)临床分布及耐药情况,为制定预防措施及指导临床合理用药提供依据。方法对文山州人民医院分离的8 820株E.coli菌株进行分离、鉴定和描述性流行病学分析;同时,对被检出的产超广谱β-内酰胺酶(extended-spectrum β-lactamase, ESBLs)E.coli菌株进行药敏试验。结果 2012年产ESBLs菌株阳性率最高,达65.40%;2014年检测出E.coli株数1 721株,占19.51%;E.coli菌株科室分布,泌尿外科最多1 961株;标本类型以中段尿为主;年龄分布集中在>45~60岁组,检出2 433株,占27.59%;药敏试验显示,产ESBLs菌株对大多数抗生素耐药率明显高于非产ESBLs菌株,对抗生素的耐药性不断发生变化。结论 E.coli菌株检出率上升,产ESBLs菌株阳性率呈现先迅速上升后有缓慢下降的趋势。E.coli耐药形势严峻,医院应加强监测及耐药性分析,严格执行抗生素使用分级制度,定期反馈临床监测数据,为医生合理使用抗生素提供科学依据。     

Nucleotide sequence of Pseudomonas aeruginosa conjugative plasmid pUM505 containing virulence and heavy-metal resistance genes

We determined the complete nucleotide sequence of conjugative plasmid pUM505 isolated from a clinical strain of Pseudomonas aeruginosa. The plasmid had a length of 123,322 bp and contained 138 complete coding regions, including 46% open reading frames encoding hypothetical proteins. pUM505 can be considered a hybrid plasmid because it presents two well-defined regions. The first region corresponded to a larger DNA segment with homology to a pathogenicity island from virulent Pseudomonas strains; this island in pUM505 was comprised of genes probably involved in virulence and genes encoding proteins implicated in replication, maintenance and plasmid transfer. Sequence analysis identified pil genes encoding a type IV secretion system, establishing pUM505 as a member of the family of IncI1 plasmids. Plasmid pUM505 also contained virB4/virD4 homologues, which are linked to virulence in other plasmids. The second region, smaller in length, contains inorganic mercury and chromate resistance gene clusters both flanked by putative mobile elements. Although no genes for antibiotic resistance were identified, when pUM505 was transferred to a recipient strain of P. aeruginosa it conferred resistance to the fluoroquinolone ciprofloxacin. pUM505 also conferred resistance to the superoxide radical generator paraquat. pUM505 could provide Pseudomonas strains with a wide variety of adaptive traits such as virulence, heavy-metal and antibiotic resistance and oxidative stress tolerance which can be selective factors for the distribution and prevalence of this plasmid in diverse environments, including hospitals and heavy metal contaminated soils.     

Detection of ampicillin and tetracycline resistance genes in uropathogenic Escherichia coli strains by affinity-based nucleic acid hybrid collection

Abstract Uropathogenic Escherichia coli strains were analyzed for the presence of the ampicillin (Ap) and tetracycline (Tc) resistance genes by a novel nucleic acid solution hybridization technique. In this method probe pairs subcloned from the resistance genes are utilized. DNA from single colonies of 20 E. coli strains was released and analyzed without purification by the 3 h hybridization test. The respective resistance genes were easily identified by the test.     

Characterization of the Escherichia coli O59 and O155 O-antigen gene clusters: the atypical wzx genes are evolutionary related

O-antigens are highly polymorphic. The genes specifically involved in O-antigen synthesis are generally grouped together on the chromosome as a gene cluster. In Escherichia coli, the O-antigen gene clusters are characteristically located between the housekeeping genes galF and gnd. In this study, the O-antigen gene clusters of E. coli O59 and E. coli O155 were sequenced. The former was found to contain genes for GDP-mannose synthesis, glycosyltransferase genes and the O-antigen polymerase gene (wzy), while the latter contained only glycosyltransferase genes and wzy. O unit flippase genes (wzx) were found immediately downstream of the gnd gene, in the region between the gnd and hisI genes in these two strains. This atypical location of wzx has not been reported before, and furthermore these two genes complemented in trans despite the fact that different O-antigen structures are present in E. coli O59 and O155. A putative acetyltransferase gene was found downstream of wzx in both strains. Comparison of the region between gnd and hisI revealed that the wzx and acetyltransferase genes are closely related between E. coli O59 and O155, indicating that the two gene clusters arose recently from a common ancestor. This work provides further evidence for the O-antigen gene cluster having formed gradually, and selection pressure will eventually bring O-antigen genes into a single cluster. Genes specific for E. coli O59 and O155, respectively, were also identified.