Impact of Genetic Diversity of Uropathogenic Escherichia coli and Klebsiella pneumoniae Strains on the Dissemination of Extended Spectrum Beta-Lactam Resistance Genes in Côte d’Ivoire

The increase and spread of bacterial resistance to extended-spectrum beta-lactam antibiotics are reported in many infections and are a real public health problem worldwide. Drug pressure is a factor that favors the emergence of a population of better adapted bacteria. However, there is no literature highlighting the genetic diversity and evolutionary structure of E. coli and K. pneumoniae in an environment with high selection pressure in Côte d’Ivoire. The objective of this study was to evaluate the genetic diversity of E. coli and K. pneumoniae strains circulating at the HKB Hospital in Abobo and at the Daloa Regional Hospital and its impact on the dissemination of extended spectrum beta-lactam resistance genes. A total of 39 strains isolated from the urinary tract of infected patients, including 30 strains of E. coli and 9 strains of K. pneumoniae were studied. A total of 39 strains isolated from the urinary tract of infected patients, including 30 strains of E. coli and 9 strains of K. pneumoniae were studied. From genomic DNA extracts, ESBL resistance genes were amplified by PCR and sequenced, in addition to genetic typing by ERIC-PCR. The data obtained were submitted to genetic and bioinformatics analyses. The results have shown a genetic diversity important in E. coli and K. pneumoniae with diversity indexs (SID) ranging from 0.5 to 0.77. The genetic structure of the bacterial species studied has shown a clonal distribution of strains with clones expressing TEM-9 and CTX-M-15 variants. Also, this clonal structure was correlated with the spread of resistance genes in E. coli and K. pneumoniae. The spread of resistant clones is a factor that might limit the fight against antibiotic resistance.

Characterization of a bla_(CTX-M-3),bla_(KPC-2)and bla_(TEM-1B)co-producing IncN plasmid in Escherichia coli of chicken origin

An extensively drug-resistant(XDR)Escherichia coli strain 258E was isolated from an anal swab sample of a chicken farm of Anhui province in China.Genomic analyses indicated that the strain 258E harbors an incompatibility group N(IncN)plasmid pEC258-3,which co-produces bla_(CTX-M-3),bla_(KPC-2),bla_(TEM-1B),qnrS1,aac(6')-Ib-cr,dfrA14,arr-3,and aac(6')-Ib3.Multiple genome arrangement analyses indicated that pEC258-3 is highly homologous with pCRKP-1-KPC discovered in Klebsiella pneumoniae from a patient.Furthermore,conjugation experiments proved that plasmid pEC258-3 can be transferred horizontally and may pose a significant potential threat in animals,community and hospital settings.

Destruction of <i>Escherichia coli</i>and Broad-Host-Range Plasmid DNA in Treated Wastewater by Dissolved Ozone Disinfection under Laboratory and Field Conditions

Broad-host-range plasmids are frequently associated with antibiotic resistance genes and can quickly spread antibiotic resistant phenotypes among diverse bacterial populations. Wastewater treatment plants have been identified as reservoirs for broad-host-range plasmids carrying resistance genes. The threat of broad-host-range plasmids released into the environment from wastewater treatment plants has identified the need for disinfection protocols to target broad-host- range plasmid destruction. Here we evaluate the efficacy of dissolved ozone at 2 and 8 mg·L–1 as a primary means for the destruction of broad-host-range plasmid and chromosomal DNA in simulated effluent. Pilot-scale tests using an experimental unit were carried out in municipal wastewater treatment plant effluent and compared with ultraviolet (UV)-irradiation and chlorination methodologies. Genes specific to Escherichia coli (uidA) and IncP broad-host-range plasmids (trfA) were monitored using real-time quantitative polymerase chain reaction (qPCR), and total DNA was monitored using absorbance spectroscopy. In wastewater treatment plant experiments, E. coli qPCR results were compared to a recognized culture-based method (Colilert?) for E. coli. In laboratory experiments, dissolved ozone at 8 mg·L–1 significantly destroyed 93% total, 98% E. coli, and 99% of broad-host-range plasmid DNA. Ozonation, UV-irradiation, and chlorination significantly reduced DNA concentrations and culturable E. coli in wastewater treat- ment plant effluent. Chlorination and UV disinfection resulted in 3-log decreases in culture-based E. coli concentrations in wastewater treatment plant effluent while changes were not significant when measured with qPCR. Only ozonation significantly decreased the IncP broad-host-range plasmid trfA gene, although concentrations of 2.2 × 105 copies trfA·L–1 remained in effluent. Disinfection processes utilizing high dissolved ozone concentrations for the destruction of emerging contaminants such as broad-host-range plasmid and total DNA may have utility as methods to ensure downstream environmental health and safe water reuse become more important.

Multidrug Resistant Pattern and Plasmid Detection of Escherichia coli from Various Sources within the University of Port Harcourt

Multi-drug resistance (MDR) in Enterobacteriaceae poses critical public health threat in Nigeria and the global world. This resistant mechanism might be plasmid mediated or chromosomal. Escherichia coli are Gram negative pathogen with a global distribution rate. The study was carried out to determine MDR and plasmid profiling of E. coli isolates from urine, feaces and poultry litter. The samples were cultured on eosine methylene blue agar and incubated for 24 hours at 37&#176;C. Results obtained showed a percentage prevalence of 30% for the urine samples which were the most prevalent, while the prevalence of E. coli from the feacal and poultry litter was 8% and 28% respectively. Identified E. coli were screened for antibiotic susceptibility by Kirby Bauer diffusion method. The results on susceptibility of E. coli to tested antibiotics before plasmid curing showed 100% resistance to cefuroxime and augumentin, while 75% resistance was observed in gentamicine, ciprofloxacin and ofloxacine. Cefixime and cefdazidime resistance were 62.5% on E. coli and the least resistance was observed in nitrofurantion (25%). The poultry litter and urine isolates recorded lower resistance level to antibiotics, compared to the feacal isolates. After plasmid curing the percentage of resistance reduced. The only antibiotics that responded positively was nitrofurantion, with high sensitivity of 87% for feacal isolate, 100% for urine isolates, and 78% for poultry litter isolates after plasmid curing. Twenty (20) of the thirty seven (37) isolates were still resistant to more than two antibiotics after the plasmid curing. Of the twenty isolates, 18 (90%) were found to harbor single plasmid, while 2 (10%) did not possess plasmid. This study concludes that nitrofurantion was the most effective antibiotics on Escherichia coli and plasmids were responsible partly for resistance.

Genetic Diversity of Uropathogenic Escherichia coli Isolated from Human T Lymphotropic Virus Type I (HTLV-I) Infected Individuals

Urinary tract infection (UTI) is a frequent pathology among HTLV-I+ individuals being capable of severely compromising the kidneys and bladder. Molecular characteristics of uropathogenic Escherichia coli (UPEC) from HTLV-I+ infected individuals are unknown. UPEC isolates from HTVL-I+ individuals, with and without clinical symptoms of myelopathy, were submitted to genetic typing seeking to infer bacterial diversity and potential virulence. 71 bacterial isolates were characterized according to random amplified polymorphic DNA and phylotypes. Phylotyping classified E. coli into four phylogenetic groups: A (18.3%), B1 (16.9%), B2 (39.4%), and D (25.3%) and 8 phylotypes according to the presence of the genetic sequences chuA, yjaA and the DNA fragment TSPE4.C2: ﹣﹣﹣ (5.6%) and ﹣+﹣ (12.6%) in phylogroup A, ﹣﹣+ (7.0%) and ﹣++ (9.8%) in B1, +++ (32.3%) and ++﹣ (7.0%) in B2, +﹣﹣ (15.4%) and +﹣+ (9.8%) in D. The B2 phylogroup was the most prevalent among HTLV﹣ associated myelopathy and asymptomatic individuals. RAPD-PCR typing revealed a high degree of bacterial polymorphism indicating a non-clonal origin. Genotypes were not found to be distributed according to clinical status or epidemiological features. Our results lead us to suggest that the neurological impairment in HTLV-I+ individuals can be a risk factor for urinary infections due E. coli which are caused by distinct bacterial lineages.

Optimization of the bioconversion of glycerol to ethanol using Escherichia coli by implementing a bi-level programming framework for proposing gene transcription control strategies based on genetic algorithms

In silico approaches for metabolites optimization have been derived from the flood of sequenced and annotated genomes. However, there exist still numerous degrees of freedom in terms of optimization algorithm approaches that can be exploited in order to enhance yield of processes which are based on biological reactions. Here, we propose an evolutionary approach aiming to suggest different mutant for augmenting ethanol yield using glycerol as substrate in Escherichia coli. We found that this algorithm, even though is far from providing the global optimum, is able to uncover genes that a global optimizer would be incapable of. By over-expressing accB, eno, dapE, and accA mutants in ethanol production was augmented up to 2 fold compared to its counterpart E. coli BW25113.

Why the discovery of adherent-invasive Escherichia coli molecular markers is so challenging?

Adherent-invasive Escherichia coli(AIEC)strains have been extensively related to Crohn’s disease(CD)etiopathogenesis.Higher AIEC prevalence in CD patients versus controls has been reported,and its mechanisms of pathogenicity have been linked to CD physiopathology.In CD,the therapeutic armamentarium remains limited and non-curative;hence,the necessity to better understand AIEC as a putative instigator or propagator of the disease is certain.Nonetheless,AIEC identification is currently challenging because it relies on phenotypic assays based on infected cell cultures which are highly time-consuming,laborious and non-standardizable.To address this issue,AIEC molecular mechanisms and virulence genes have been studied;however,a specific and widely distributed genetic AIEC marker is still missing.The finding of molecular tools to easily identify AIEC could be useful in the identification of AIEC carriers who could profit from personalized treatment.Also,it would significantly promote AIEC epidemiological studies.Here,we reviewed the existing data regarding AIEC genetics and presented those molecular markers that could assist with AIEC identification.Finally,we highlighted the problems behind the discovery of exclusive AIEC biomarkers and proposed strategies to facilitate the search of AIEC signature sequences.

Characteristics of colistin-resistant Escherichia coli from pig farms in Central China

The emergence and dissemination of colistin resistance in Enterobacterioceae mediated by plasmid-borne mcr genes in recent years now pose a threat to public health.In this study,we isolated and characterized colistin-resistant and for mcr-positive£coli from pig farms in Central China.Between 2018 and 2019,594 samples were collected and recovered 445 E.coli isolates.Among them,33 with colistin resistance phenotypes and 37 that were positive for mcr genes were identified,including 34 positive for mcr-1,one positive for mcr-3,and two positive for both mcr-1 and mcr-3.An insertion of nine bases("CTGGATACG")into mcr-7 in four mcr-positive isolates led to gene dysfunction,and therefore did not confer the colistin resistance phenotype.Antimicrobial susceptibility testing revealed that 37 mcr-positive isolates showed severe drug resistance profiles,as 50% of them were resistant to 20 types of antibiotics.Multilocus sequence typing revealed a heterogeneous group of sequence types in mcr-positive isolates,among which ST10(5/37),ST156(5/37),and 5T617(4/37)were the predominant types.Plasmid conjugation assays showed that mcr-carrying plasmids of 25 mcr-positive isolates were conjugated with£coli recipient,with conjugation frequencies ranging from 1.7 × 10^(-6) to 4.1 × 10^(-3) per recipient.Conjugation of these mcr genes conferred a colistin resistance phenotype upon the recipient bacterium.PCR typing of plasmids harbored in the 25 transconjugants determined six types of plasmid replicons,including lncX4(14/25),FrepB(4/25),Incl2(3/25),lncHI2(2/25),FIB(1/25),and Inch(1/25).This study contributes to the current understanding of antibiotic resistance and molecular characteristics of colistin-resistant£coli in pig farms.

幽门螺杆菌ureA、ureB基因克隆、序列分析及在E.coli中表达

目的 构建含幽门螺杆菌 (Hp) ure A、ure B基因重组质粒 ,测定、分析其核酸序列及推定的氨基酸序列 ,在 E.coli中高效表达两基因 ,为检测试剂和疫苗研究提供基础依据和抗原。方法 PCR法扩增 Hp菌株 HPSH4的 ure A、ure B基因 ,分别与 p GEX- 2 T载体连接并转化大肠杆菌JM10 5 ,测定克隆基因的核酸序列并与 Gen Bank公布的国外 5株 Hp菌株 (2 6 6 95 ,HPK 5 ,J99,CPM6 30 ,M6 0 398)的 ure A、ure B基因作序列比较 ,以 IPTG诱导 ,ure A、ure B基因在 E.coli中高效表达。结果 ure A核酸同源性 96 .3%～ 98.1% ,氨基酸同源性 98.3%～ 10 0 % ;ure B核酸同源性95 .8%～ 98.0 % ,氨基酸同源性 96 .4 %～ 99.6 %。以 IPTG诱导 ,在 E.coli中表达 rure A融合蛋白5 5 6 0 0 D,rure B融合蛋白 85 5 0 0 D。结论 不同地区 Hp菌株的 ure A、ure B存在程度不同的变异 ,克隆并表达 HPSH4的 ure A、ure B与 Gen Bank已公布的多数 Hp菌株有极高同源性 ,在 E.coli以融合蛋白高效表达 rure A和 rure

重组人肿瘤坏死因子(hTNF-α)在大肠埃希菌[E.coli BL21(DE3)]中表达条件的优化

目的构建人肿瘤坏死因子(hTNF-α)表达质粒,并对其进行鉴定,优化hTNF-α蛋白表达条件使之在大肠埃希菌中实现高效表达。方法应用PCR扩增技术获得hTNF-α基因片段并进行相应的鉴定,将鉴定后的hTNF-α基因克隆到pET24a载体中,获得pET24a-hTNF-α表达质粒。将此质粒转化入大肠埃希菌BL21(DE3)中,并对其表达条件进行优化。结果成功构建了pET24a-hTNF-α质粒,PCR和酶切技术进行鉴定,结果显示与目的片段一致。将此质粒转入大肠埃希菌BL21(DE3)中,得到最佳表达条件为:M9+LB培养基,37℃,0.5 mmol/L IPTG,pH值=7.5,诱导时间为5 h。优化后的菌体干重增高了2.56倍,hTNF-α产率增加3.68倍,hTNF-α表达率从9.38%增加到32.74%,增高了3.49倍。结论优化了pET24a-hTNF-α质粒在大肠埃希菌BL21(DE3)中的表达条件,使hTNF-α蛋白得到高效表达。

过量表达葡萄糖脱氢酶对E.coli羟基脂肪酸合成能力的影响

在工程大肠杆菌生产羟基脂肪酸的基础上,为解决构建的以葡萄糖为底物合成羟基脂肪酸(HFAs)代谢途径中存在的细胞内还原力(NADPH)不平衡的问题,克隆了来源于巨大芽孢杆菌葡萄糖脱氢酶(GDH)基因,构建了胞内HFAs和NADPH联产的工程菌株;实验结果表明该重组大肠杆菌合成HFAs的产量得到显著提高,摇瓶条件下HFAs产量达到173.9 mg/L。具有较好的工业化开发前景。

Characteristics of two transferable aminoglycoside resistance plasmids in Escherichia coli isolated from pig and chicken manure

Plasmid-mediated antibiotic resistance genes (ARGs) have recently become a more prominent concern in the global environment. However, the prevalence of aminoglycoside resistance plasmids in the livestock industry is under reported. In this study, two transferable aminoglycoside resistance plasmids, pRKZ3 and pKANJ7, isolated from pig and chicken manure, were characterized. Results showed that pRKZ3 (8236 bp) is a non-conjugative IncQ plasmid and contains genes encoding for plasmid replication and stabilization (repA, repB and repC), mobilization (mob), and antibiotic resistance (arr-3 and aacA). pKANJ7 (30142 bp) is a conjugative IncX plasmid which codes for a type IV secretion system (T4SS). Conjugative transfer experiments showed that the optimal mating time of pKANJ7 was 8 h under the starvation condition, but the number of tranconjugants increased with time under the nutrient condition. Statistical analysis indicated that the two plasmids had little impact on the growth of their hosts, but a relatively high level of fitness cost due to pKANJ7 was observed. We also found that the fitness cost of plasmids depended on their hosts. Compared with pKANJ7, the relative fitness cost index of pRKZ3 varied within a narrow range during the 10 days of competition. The low level of fitness cost of pRKZ3 might contribute to the persistence of the plasmid in the environment. Our study provides new information for understanding the characterizations of antibiotic resistance plasmids (ARPs) in manure sources and helps to clarify the transfer and persistence of ARPs in the environment following the application of manure.

Preparation of Conotoxin MrVIB by Genetic Engineering Technology

[ Objective] The disulfide-rich conotoxin MrV1B was produced by simple and fast genetic engineering method, to find new efficient ways for the synthesis of natural active conotoxins. [Method] Primers of conotoxin gene MrVIB were synthesized to construct expression vectors pET22b（ ＋ ）/His-Xa-MrVIB and pET32a/Trx-EK-MrV1B, which were transformed into BL21 （DE3）pLysS and expressed under induction by IPTG. Recombinant proteins were purified by affinity chromatography using Ni-NTA agarose column, and the expression of the recombinant proteins was analyzed by Tricine-SDS-PAGE electrophoresis. [ Result] The recombinant conotoxins His-Xa-MrVIB and Trx-EK-MrVIB were effectively expressed in E. coli, and purified by one-step affinity chromatography, and the purity of the recombinant conotoxins was greater than 90%. [ Conclusion] The conotoxin MrVIB was effectively secreted and expressed by genetic engineering method, which could solve the problems in chemical synthesis of conotoxins including low yield, high cost and difficult purification.

High-level expression of foreign genes via multiple joined operons and a new concept regarding the restricted constant of total amount of plasmid DNA per Escherichia coli cell

OBJECTIVE: To examine the feasibility of linking operons in tandem to enhance expression of heterologous genes in Escherichia coli (E. coli) and clarify the potential control mechanism of the total plasmid DNA amount in each host cell. METHODS: Two series of expression plasmids, CW11 and CW12, containing 1 to 4 and 1 to 3 heterologous gene operon(s) respectively, were constructed. The molecular size of the CW11 series varied from 5.47 kb to 12.26 kb in 2.25 kb increments. The CW12 series varied from 5.40 kb to 9.72 kb in 2.16 kb increments. The expression level of desired protein was assayed by SDS-PAGE and laser density scanning. Plasmid copy number was determined by incorporation with (3)H-thymidine ((3)H-TdR). RESULTS: No influence of the tandem-joined operons on host growth and plasmid stability was observed. Upon induction, the desired protein accumulations in the CW11 series were 44.9% +/- 3.9%, 51.3% +/- 4.1%, 54.8% +/- 3.3% and 58.2% +/- 3.4% of total cell protein. In the CW12 series, the yields were 32.2% +/- 5.0%, 42.8% +/- 4.1% and 46.9% +/- 4.0% of total cell protein. As size increased, the plasmid copy number decreased, but target gene dosage increased significantly (P 0.05) and restricted to some extent. CONCLUSIONS: Increasing the target gene dosage by tandem linking of operons may enhance the expression level of a desired protein. Although the size (kb) and the copy number of each plasmid are negatively interrelated, for certain plasmids in each series, their total DNA amount per cell seems to be a restricted constant for specific E. coli strains under identical incubation condition.

携带bla_(CTX-M)禽大肠杆菌的多重耐药特征和体外致病性研究

【目的】探究携带bla_(CTX-M)禽大肠杆菌的分子流行特点。【方法】检测76株携带bla_(CTX-M)禽大肠杆菌的多重耐药谱、系统进化分群及毒力基因,并分析毒力基因与耐药特点、系统进化分群之间的关联性。【结果】药敏结果显示,携带bla_(CTX-M)禽大肠杆菌对氟苯尼考的耐药率最高,达85.53%(65/76);其次是恩诺沙星,为48.68%(37/76);最常见的多重耐药谱为氟苯尼考+恩诺沙星+乙酰甲喹,达10.53%(8/76)。系统进化分群结果显示,主要的亚群为C群和B1群;其中,C群检出率最高,达53.95%(41/76);其次是B1群,为30.26%(23/76);E群、A群、D群和F群的检出率分别为9.21%(7/76)、2.63%(2/76)、2.63%(2/76)和1.32%(1/76);未检测出B2群和分支I群。毒力基因检测结果显示,76株携带bla_(CTX-M)禽大肠杆菌均检出了至少4种毒力因子,且有11株菌同时携带至少11种毒力基因;其中,有13株菌同时检出禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)的5个标志性毒力基因(iroN、iutA、hly、OmpT和iss),检出率为17.11%;有11株菌检出ColV质粒的标志基因(cva/cvi、iroN、iucD/iutA、etsAC、OmpT/hlyF和sitA),检出率为14.47%;有28株菌检出耶尔森菌强毒力岛(high pathogenitility island,HPI)的标志基因irp-2和fyuA,检出率为36.82%。【结论】携带bla_(CTX-M)禽大肠杆菌多为多重耐药菌,且多重耐药菌株携带ColV毒力质粒可能性更高;携带bla_(CTX-M)禽大肠杆菌的多重耐药与致病性有关联,即携带bla_(CTX-M)禽大肠杆菌具有多重耐药的同时,具有致病的可能性更高。

一种诱导表达绿色荧光蛋白穿梭质粒的构建及其在荧光示踪中的应用

细菌的荧光标记是一种重要的常用实验标记技术,用于研究细菌生理生化特性、感染宿主体内示踪、感染细胞示踪等研究。细菌的荧光标记通常通过质粒表达荧光蛋白来实现,而不同类型的细菌,由于菌种特性不同,构建的质粒往往不能通用。本研究构建了一种可诱导表达增强型绿色荧光蛋白的穿梭质粒pBT-iEGFP,该质粒可通过添加无水四环素诱导表达绿色荧光蛋白。诱导表达和传代稳定性试验表明,pBT-iEGFP质粒可在禽致病性大肠杆菌、鼠伤寒沙门菌和马耳他布鲁菌中成功诱导表达绿色荧光蛋白,在五代盲传中该质粒能在细菌中稳定遗传。胞内布鲁菌诱导表达试验证实,pBT-iEGFP质粒可成功示踪细胞感染过程中活的布鲁菌。总之,本研究构建的pBT-iEGFP质粒可作为细菌的荧光示踪研究工具,应用于多种实验研究。

广东省腹泻仔猪大肠杆菌blaCTX-M-65与mcr-1基因共传播特征

【目的】调查分析广东省某生猪养殖场腹泻仔猪的大肠杆菌耐药情况以及同时携带blaCTX-M-65与mcr-1两种耐药基因的阳性大肠杆菌的分子特征与其共同传播特征,为耐药性监测及风险防控提供科学依据。【方法】从广东省肇庆市某生猪养殖场采集腹泻仔猪直肠棉拭子样品90份,并进行大肠杆菌分离鉴定;采用PCR方法检测产超广谱β-内酰胺酶(ESBLs)与mcr-1基因,通过测序确定CTX-M亚型;采用琼脂二倍稀释法和微量肉汤稀释法测定分离菌对12种抗菌药物的最小抑菌浓度(MIC);使用脉冲场凝胶电泳(PFGE)及多位点序列分型(MLST)分析菌株间的遗传背景;通过接合转移方法确定质粒水平转移的能力;使用复制子分型、S1-PFGE、Southern blotting方法检测质粒类型、耐药基因的定位及定位质粒的大小;通过三代测序及序列分析确定质粒的重组过程。【结果】共分离鉴定86株大肠杆菌,检测到44株携带CTX-M型ESBLs基因,其中blaCTX-M-55基因最流行(n=16,36.4%),其次是blaCTX-M-65(n=10,22.7%)、blaCTX-M-27(n=8,18.2%)、blaCTX-M-15(n=5,11.4%),还检测出blaCTX-M-79(n=2,4.5%)、blaCTX-M-14(n=2,4.5%)和blaCTX-M-24(n=1,2.3%)。10株blaCTX-M-65基因阳性菌有8株同时携带mcr-1基因。这8株大肠杆菌均表现为多药耐药,包括氨苄西林、阿莫西林、头孢噻呋、头孢喹肟、黏菌素等多种抗生素。8株菌共分为6种ST型、7个PFGE谱型。接合转移试验发现,有6株菌的blaCTX-M-65和mcr-1基因发生了共转移,且blaCTX-M-65与mcr-1基因均位于IncHI2型(253 kb)质粒上。其中1株菌在接合转移的过程中,其所携带的一个253 kb IncHI2质粒与一个69 kb IncFⅡ质粒发生融合,形成一个大小323 kb的融合质粒。对融合质粒进行三代测序解析,结果表明,在接合转移的过程中IS26介导了两个不同大小质粒的重组。【结论】IncHI2质粒是介导blaCTX-M-65和mcr-1基因共同传播的主要媒介,IS26通过与靶位点GTTTCACT的整合导致IncHI2质粒与IncFⅡ质粒融合。质粒重组扩大了大肠杆菌的耐药谱,加速了耐药基因的传播,促使多药耐药现象更严重,对公共卫生安全构成威胁,本研究为阐明多重耐药大肠杆菌的传播机制提供了参考依据。

首次从湖南猪源大肠杆菌中检出mcr-1阳性IncI2(Delta)质粒

近年来,由于抗生素耐药基因的广泛传播及多重耐药细菌的出现,导致抗生素的使用面临严峻挑战,致使毒性较强的多黏菌素又重新引起业界的重视,为此对多黏菌素耐药情况的研究也变得十分重要。为了调查畜牧场中多黏菌素耐药基因mcr-1的流行情况,本研究选择我国湖南省某猪场240份肠肛拭子样品,培养分离鉴定出含有mcr-1耐药基因的大肠杆菌菌株,选取多黏菌素等10种抗菌药物对分离的菌株进行耐药性试验,对所有含mcr-1耐药基因的大肠杆菌阳性株进行全基因组测序(whole-genome sequencing,WGS),采用多位点序列分型(multi-locus sequence typing,MLST)技术进行大肠杆菌的遗传多样性分析。研究结果表明,在分离出的172株大肠杆菌中,来自不同个体的9株携带mcr-1基因的大肠杆菌均表现出多重耐药现象;MLST分析共鉴定出ST10、ST196、ST46和ST5229共4种分型和4种血清型(O83:H5,O16:H7,O16:H51,O9:H4)。生物信息学分析显示,所有阳性菌株均未发现与mcr-1基因传播有关的典型可移动遗传元件ISApl1,但均检出可能会导致mcr-1传播的IV型分泌系统基因。质粒接合转移试验与单倍型的MLST多态性结果表明,mcr-1基因主要以质粒介导的水平传播为主。本研究是国际上首次在猪源细菌中检出常见于人医临床细菌的携带有mcr-1抗性基因的IncI2(Delta)质粒。

猪肠道非致病性且无耐药性大肠杆菌的分离鉴定、生长特性和基因组进化分析

【目的】从猪肠道中分离出无毒力基因且无抗生素耐药性的大肠杆菌,并分析其生长性能和遗传进化规律,以期为开发猪用微生态制剂或口服疫苗奠定基础。【方法】采集广东省2个地区养猪场的11头和15头健康成年猪的粪便样本。首先,采用培养方法从麦康凯培养基中分离出疑似大肠杆菌的菌株,通过16S rRNA序列分析鉴定其是否为大肠杆菌菌株;然后,采用PCR方法验证所选大肠杆菌是否含有13种常见的猪致病性大肠杆菌毒力基因,并以7类(14种)抗生素进行药敏试验;最后,对筛选出的优质大肠杆菌菌株进行生长动力学分析,并提取其全基因组DNA,构建全基因组系统进化树。【结果】在260株疑似为大肠杆菌菌株中,经16S rRNA序列分析确认后有206株属于大肠杆菌。其中,107株大肠杆菌不含13种常见毒力基因,25株大肠杆菌对7类(14种)抗生素敏感。在这25株菌株中,有23株非致病性且无抗生素耐药性大肠杆菌(编号为2-9、3-2、3-4、5-1、6-1、6-2、6-9、6-10、8-2、8-9、10-5、B-4、B-6、B-7、B-10、D-10、E-2、E-10、J-1、J-4、K-6、L-6和O-9)的生长性能与大肠杆菌Nissle1917、MG1655相似,其他2株大肠杆菌(编号为6-4和5-2)的生长速度高于Nissle 1917和MG1655。此外,有10株菌株(编号为E-10、E-2、6-4、6-9、8-2、O-9、3-2、6-2、J-1、K-6)耐酸性能较好,可能适合长期定殖于猪胃肠道内。基于SNP核心基因组系统进化树分析,发现这25株菌株中有9株菌株(编号为3-2、6-10、10-5、6-1、8-9、5-2、L-6、O-9、K-6)在系统进化树开端处属于同一大类群,表明它们极可能为猪原生大肠杆菌的祖先群。【结论】综合上述大肠杆菌的抗生素敏感性、生长性能和全基因组进化树和胃液环境耐受性能分析,菌株O-9在这4个方面均具备优势,该菌株与大肠杆菌Nissle 1917、MG1655的生长动力学相似甚至更优,意味着O-9菌株可能是优质的猪肠道原生大肠杆菌,具有极大研究潜力。

pDNA质粒在一次性生物反应器中的放大生产研究

【目的】探索大肠杆菌(E.coli)菌种复苏后跳过三角摇瓶直接接种一次性WAVE生物反应器进行菌种扩增,并放大至一次性XDR生物反应器进行质粒放大生产的可行性,建立GMP级质粒在全一次性上游平台进行工业化放大生产的工艺。【方法】通过碳氮比优化,筛选并获得不含任何动物源成分的基础培养基和补料培养基;菌种冻存管室温融化后以低密度接种三角摇瓶和WAVE反应器,考查菌种低密度接种的可行性,比较三角摇瓶和WAVE反应器进行菌种扩增的差异,建立菌种在WAVE反应器中的扩增工艺;随后将菌种扩增至50LXDR生物反应器进行质粒的放大生产。【结果】与LB培养基相比,优化的不含任何动物源成分的基础培养基使菌体最高密度和质粒产量分别提高43%和77%;以1∶1000-1∶8000低密度接种WAVE反应器,菌种比生长速率达到(0.65±0.065)/h,WAVE反应器展现了更好的过程参数控制,通过一级WAVE种子罐可直接为50-200 L生产罐提供种子细胞;质粒在一次性50LXDR反应器中的放大生产,最高菌体密度和质粒产量分别达到53OD和340mg/L,质粒比生产速率达到6.42 mg/L/OD_(600),比常规质粒比生产速率提高2倍以上,超螺旋质粒比例达到90%,较高的上游收获超螺旋比例为下游质粒两步层析纯化提供了可能。【结论】建立了大肠杆菌通过WAVE反应器进行菌种扩增,通过一级种子罐直接接种生产罐进行质粒放大生产的工艺,为GMP级别质粒在50-200L一次性生产平台中的放大生产建立了生产工艺。