Prevalence of Escherichia coli O157:H7 among diarrhoeic HIV/AIDS patients in the Eastern Cape Province-South Africa

This study investigated the prevalence of Escherichia coli O157:H7 in the stool of confirmed and non-confirmed diarrhoeic HIV/AIDS patients. Escherichia coli O157:H7 was isolated by culture-based and immunomagnetic separation from three hundred and sixty stool swabs. Identification was by conventional IMViC, 20E API and molecular techniques. Confirmed and non-confirmed diarrhoeic HIV/AIDS patients had 56.5% (74/131) and 43.5% (57/131) respectively of E. coli O157:H7. Molecular results indicated that the prevalence of E. coli O157:H7 was 12.16% (9/74) and 8.77% (5/57) from stool swabs of confirmed and non-confirmed diarrhoeic HIV/AIDS patients. Antimicrobial resistance was higher for E. coli O157:H7 isolates from stools of confirmed HIV/AIDS than it was for non-confirmed HIV/AIDS patients. Escherichia coli O157:H7 might be a silent cause of diarrhoea in HIV/AIDS patients. It is recommended that HIV/AIDS patients with diarrhoea should be screened for E. coli O157:H7 and surveillance programmes for these bacteria should be established in both urban and rural areas of South Africa.     

Chitosan-Based Intranasal Vaccine against Escherichia coli O157:H7

Background:

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an infectious zoonotic pathogen causing human infections. These infections, in some cases, can lead to hemolytic uremic syndrome and its life-threatening complications and even death worldwide. The first intimate bacterial adhesion, intimin (I), with its own receptor translocated intimin receptor (Tir) and E. coli secreted protein A, acting as Tir conduit, are highly immunogenic proteins for vaccine development against E. coli O157:H7.

Methods:

A chimeric trivalent recombinant protein was previously found to be a suitable strategy for developing vaccines against E. coli O157:H7. In this study, the recombinant EIT (rEIT) was used to design a protective EHEC nasal nanovaccine. Chitosan and its water-soluble derivative, trimethylated chitosan (TMC), as muco-adhesive biopolymers, are good candidates for preparation of nanovaccines.  Using the electrospraying technique, as a novel method, we could obtain particles of rEIT loaded with chitosan and TMC on a nanometer scale. Mice were immunized with intranasal administration or intrapretoneal injection of rEIT.

Results:

The rEIT-specific immune responses (IgG and IgA) were measured by indirect ELISA. Only nasal administration of chitosan electrospray and TMC formulation produced significant secretion IgA. Intranasal administration of nanovaccine reduced the duration of bacterial fecal shedding on mice challenged with E. coli O157:H7.

Conclusion:

Since development of mucosal vaccines for the prevention of infectious diseases requires efficient antigen delivery; therefore, this research could be a new strategy for developing vaccine against E. coli O157:H7.Key Words: EnterohemorrhagicEscherichia coli, Nanoparticles, Intranasal vaccination     

Controlled expression of cholera toxin B subunit from Vibrio cholerae in Escherichia coli

The ctxB gene, the causative agent of cholera epidemic was successfully cloned from V. cholerae in E. coli. The insertion of the gene was confirmed by PCR as well as restriction digestion analyses. The sequencing results for the gene confirmed that the insert was in the correct orientation and in-frame with the P(BAD) promoter and it showed that the gene was 99% homologous to the published ctxB sequence. The CTB protein was successfully expressed in E. coli using the pBAD/His vector system. The expected protein of approximately 14 kDa was detected by SDS-PAGE and Western blot. The use of pBAD/His vector to express the cholera toxin gene in E. coli would facilitate future study of toxin gene products.     

短短芽胞杆菌FJAT-0809-GLX超氧化物歧化酶基因的克隆及原核表达

短短芽胞杆菌FJAT-0809-GLX具有抑菌、抗褐变和保鲜功能。以短短芽胞杆菌菌株FJAT-0809-GLX总DNA为模板,采用PCR扩增超氧化物歧化酶(Supseroxide dismutase,SOD)基因,将该片段纯化回收后与p MD18-T连接并转入大肠杆菌(Escherichia coli)DH5α,进行序列测定,结果显示SOD基因序列长度为609 bp(Gen Bank登录号:KM255665),编码202个氨基酸残基。将SOD基因片段与同样酶切的表达载体p ET-28a连接,构建重组表达载体p ET-28a-SOD,转入大肠杆菌BL-21,采用异丙基-β-D-硫代吡喃半乳糖苷(Isopropyl-β-D-thiogalactopyranoside,IPTG)进行诱导表达。SDS-PAGE结果表明,在菌体中存在约25 ku的蛋白表达产物,与该基因ORF预期大小接近。以上结果为进一步研究该酶的生理生化特性奠定了基础。     

Durable antimicrobial cotton fabrics treated with a novel N-halamine compound

5,5-Dimethyl-3-((3’-triethoxysilylpropylamido)propyl)hydantoin (Si-Hy), a novel N-halamine precursor, has been synthesized in this work. The traditional pad-dry-cure process was used to coat the produced Si-Hy onto cotton fabrics. The coated fabric was characterized by SEM, FTIR and XPS. After exposure to chlorine bleach, the treated fabric presented good antimicrobial ability. The chlorinated sample demonstrated potent antibacterial ability against S. aureus (ATCC 6538) and E. coli O157:H7 (ATCC 43895) in brief contact time. Sixty seven percent of oxidative chlorine was retained and over 85 % of chlorine could be recharged after storage for 15 days and rechlorination. The antibacterial materials with good biocidal efficacies have potential applications in the healthcare industry.     

茶氨酸生物合成工程菌构建

通过PCR扩增E.coli DH5α的γ-ggt基因,产物经纯化后用Kpn I和Xho I双酶切,回收γ-谷氨酰转肽酶基因目的片断,并与经相同双酶切的表达载体pET-32a连接,得到重组质粒pET-GGT。将重组质粒转化到E.coli BL21中,获得工程菌。工程菌株经0.05βmol/L IPTG,32℃诱导表达,湿菌体的酶活达到2.0βU/g,大约是出发菌株E.coli DH5α的15倍。工程菌催化L-谷氨酰胺和盐酸乙胺反应生成茶氨酸的产量达到29.40βg/L,L-Gln的转化率为48.22%,其催化L-谷氨酰胺和盐酸乙胺反应生成茶氨酸的能力比出发菌株E.coli DH5α提高了100多倍。     

Immunogenicity of a Fusion Protein Comprising Coli Surface Antigen 3 and Labile B Subunit of Enterotoxigenic Escherichia coli

Background: Enterotoxigenic Escherichia coli (ETEC) strains are the major causes of diarrheal disease in humans and animals. Colonization factors and enterotoxins are the major virulence factors in ETEC pathogenesis. For the broad-spectrum protection against ETEC, one could focus on colonization factors and non-toxic heat labile as a vaccine candidate. Methods: A fusion protein is composed of a major fimbrial subunit of coli surface antigen 3, and the heat-labile B subunit (LTB) was constructed as a chimeric immunogen. For optimum level expression of protein, the gene was synthesized with codon bias of E. coli. Also, recombinant protein was expressed in E. coli BL21DE3. ELISA and Western tests were carried out for determination of antigen and specificity of antibody raised against recombinant protein in animals. The anti-toxicity and anti-adherence properties of the immune sera against ETEC were also evaluated. Results: Immunological analyses showed the production of high titer of specific antibody in immunized mice. The built-in LTB retains native toxin properties which were approved by GM₁ binding assay. Pre-treatment of the ETEC cells with anti-sera significantly decreased their adhesion to Caco-2 cells. Conclusion: The results indicated the efficacy of the recombinant chimeric protein as an effective immunogen inducing strong humoral response. The designated chimer would be an interesting prototype for a vaccine and worthy of further investigation. Key Words: Recombinant vaccine, Enterotoxigenic Escherichia coli (ETEC), cstH, eltB     

齿兰环斑病毒外壳蛋白基因的表达及其表达产物抗血清的制备

用pET－21d构建齿兰环斑病毒（ORSV）外壳蛋白基因大肠杆菌表达载体pEO。经SDS－PAGE和Westernblotting分析，含pEO的大肠杆菌正确表达了ORSV外壳蛋白基因。此表达产物为融合蛋白。用表达产物制备抗血清，并应用于间接酶联法检测ORSV，具有较高的灵敏度（10ng／mLORSV）和特异性。     

茶树花青素还原酶基因在大肠杆菌中的表达及优化

茶树花青素还原酶是催化非酯型儿茶素EC和EGC合成的关键酶。采用RT-PCR技术,获得了茶树花青素还原酶基因的开放阅读框,它编码含337个氨基酸的蛋白质,推测分子量为37kD,等电点为6.54;成功地将该基因重组到表达载体pET32a(+)上,并在大肠杆菌rosetta中进行原核表达;优化了原核表达中诱导时间、诱导温度、IPTG浓度、氨苄青霉素浓度,纯化出目的蛋白。HPLC检测表明,目的蛋白具有ANR酶活性。     

N-halamine-bonded cotton fabric with antimicrobial and easy-care properties

N-halamine precursor 2,2,6,6-tetramethyl piperidinol (TMP), a hindered amine light stabilizer, was bonded onto cotton fabric by using 1,2,3,4-butanetetracarboxylic acid (BTCA) as a crosslinking agent. A variety of treating conditions including TMP concentration, curing temperature and time, and catalyst were studied. The treated fabrics were characterized using FTIR spectra and scanning electron microscope (SEM). The cotton fabric treated with TMP precursor could be rendered biocidal upon exposure to dilute household bleach. The chlorinated cotton swatches showed great efficacy and inactivated 100 % of Staphylococcus aureus with 7.1 log reduction with 5 min of contact and 83.25 % of E. coli O157:H7 at 10 min of contact. In addition, the wrinkle recovery angle of the treated cotton fabrics increased from 229 ° of untreated cotton fabrics to 253 °. This study provided a practical finishing process to produce cotton fabrics with easy care and antibacterial functionalities at the same time.     

斑茅SAMDC基因的克隆及其原核表达分析

以斑茅(Erianthus arundinaceus)samdc基因的cDNA为基础,采用基因重组技术,将该基因按正确的阅读框架定向克隆于原核表达载体pET-29a(+)中,转化大肠杆菌BL21(DE3),用IPTG诱导表达,并对表达产物进行SDS-PAGE分析.结果表明:重组斑茅samdc基因在大肠杆菌中获得高效表达,其分子量约为43.281kDa.斑茅samdc基因原核表达载体的成功构建和重组斑茅SAMDC蛋白在大肠杆菌中的高效表达,为进一步研究其生物学功能奠定了基础.     

大肠杆菌glgC基因的定点突变及高效表达

通过PCR方法,从E.coliJM109基因组DNA中扩增到全长为1296bp的glgC基因,对其进行定点突变,获得第296位和336位氨基酸同时突变的glgC336基因。将2者亚克隆进pET-30a,成功构建了重组表达载体pET-C和pET-C336,转化大肠杆菌BL21(DE3),在1mmol/LIPTG诱导下进行表达。SDS-PAGE电泳分析显示,在约53kD处有1条明显的蛋白表达带,证明目的基因已得到高效表达,且重组蛋白表达量占菌体蛋白总量的77.3%。     

Antimicrobial and antioxidant activities of lignin from residue of corn stover to ethanol production

To improve the economic viability of the biofuel production from biomass resource, a value-added lignin byproduct from this process is increasingly important. Antioxidant and antimicrobial activities of lignin extracted from residue of corn stover to ethanol production were investigated. The lignin extracts exhibited strong antioxidant activities in hydrophilic oxygen radical absorbance capacity (ORAC) assay and Folin-Ciocalteu test. The extracts also exhibited antimicrobial activities against Gram-positive bacteria (Listeria monocytogenes and Staphylococcus aureus) and yeast (Candida lipolytica), but not Gram-negative bacteria (Escherichia coli O157:H7 and Salmonella Enteritidis) or bacteriophage MS2. Different extraction conditions (temperature and residue/solvent ratio) affected the antioxidant and antimicrobial activities of lignin extracts. Generally, the bioactivities of lignin extracts were consistent with FTIR analysis results. Lignin byproducts showed the potential for their antioxidant and antimicrobial application.     

Antibacterial Coating of Cellulose by Iso-bifunctional Reactive N-halamine with the Dyeing Process of Reactive Dye

In this study, we synthesized a novel N-halamine precursor, sulfuric acid mono-[2-(4-[4-chloro-6-(2-[4,4- dimethyl-2,5-dioxo-imidazolidin-1-yl]-ethylamino)-[1,3,5]triazin-2-ylamino]-benzenesulfonyl)-ethyl] ester sodium (TB), which contains two reactive groups of monochloro triazine reactive groups and bis-sulphatoethylsolphone reactive groups. The structure of TB is similar to iso-bifunctional group reactive dyes and could be coated on cotton fabrics by covalent bonds through a reactive dyeing process. The cotton coated with TB was characterized by FTIR and SEM. After chlorination, the treated cotton fabrics showed excellent antibacterial efficacy and inactivated all inoculated S. aureus (ATCC 6538) and E. coli O157: H7 (ATCC 43895) within 1 min of contact. Over 85 % of tensile strength retained both in warp and weft directions after treatment and chlorination. Almost 80 % of active chlorine can be regained by treating with household bleach after extensive washing and long time storage.     

Prevalence of Class 1 Integrons and Extended Spectrum Beta Lactamases among Multi-Drug Resistant Escherichia coli Isolates from North of Iran

Background:

Extended spectrum beta lactamases (ESBLs) are an important cause of transferable multidrug resistance (MDR) in gram-negative bacteria. The most described ESBL genes are generally found within integron-like structures as mobile genetic elements. The aim of this study was to identify the accompanying of class 1 integrons and ESBLs in the MDR E. coli isolates.

Methods:

Susceptibility to antimicrobial agents was determined for 33 E. coli strains by the disk diffusion method. Double-disk synergy test was applied for screening ESBL. To identify the strains carrying integrons, the conserved regions of integron-encoded integrase gene intI1 were amplified. For detection of gene cassettes, 5′CS and 3′CS primers were used.

Results:

All E. coli isolates were identified as multi-drug resistant. More than 50% of the isolates were resistant to tetracycline, cephalothin, cefuroxime, amoxicillin-clavulanic acid, and third generation cephalosporines. Nearly all of the isolates displayed sensitivity to piperacillin. There was a significant correlation between production of ESBL and resistance to all antibiotics except for ciprofloxacin and piperacillin (P < 0.01). Thirty two MDR strains (97%) included class 1 integron, and some isolates that included integrons were similar in the size of gene cassettes. The isolates were different in the resistance profiles; however, some others had similar resistance profiles. Of eight ESBL positive isolates, seven (87.5%) carried class 1 integrons.

Conclusion:

Class 1 integrons were frequent in MDR and also ESBL-producing E. coli isolates. High prevalence of class 1 integrons confirms that integron-mediated antimicrobial gene cassettes are important in E. coli resistance profile. Key Words: Antibiotic, Integrons, Escherichia coli     

PYH157广谱抗病基因导入高梁及转基因植株的筛选与研究

用花粉管通道法将PYH157基因导入高梁，获得了转基因植株。应用卡钠霉素和高粱丝黑穗病鉴定及聚丙烯酰胺等电聚凝胶电泳蛋白质分析表明，无毒广谱抗病基因PYH157已整合到高粱基因组中，并能表达。高粱丝黑穗病田间接种试验中筛选了4个较抗高粱丝黑穗病的转基因植株。     

Rapid detection of Salmonella enteritidis by PCR amplification of the SefA gene and it's cloning

The emergence of Salmonella enteritidis as an important food-borne pathogenesis in humans, demands the development of novel detection and intervention strategies. It is generally accepted that fimbriae are an important factor in bacterial survival and persistence in the host. This study is directed towards the method of amplifying and cloning the SefA gene, which encode Salmonella enteritidis fimbrial protein. Strains used for these studies were S. enteritidis (E3), which were collected from Kermanshah region. Chromosomal DNA was extracted by boiling method and PCR reaction was performed and single band of 511 bp amplified by SefA-F and SefA-R primers. The resulting PCR product was inserted into the cloning vector (pTZ57R/T). In order to amplify the recombinant plasmid, E. coli DH5 alpha bacteria were transformed with SefA-pTZ57R/T. Recombinant clones were identified by blue/white selection and purified recombinant plasmids were indicated by an alkaline lysis procedure. Identity of the SefA-pTZ57R/T product was confirmed by RFLP and sequencing. Nucleotide and protein alignment with BLAST software showed that the sequence of the SefA gene derived from S. enteritidis (E3), which was cloned in the pTZ57R/T vector, was 99% identical to that of the Genbank (L11008). The sequence of the SefA gene from S. enteritidis (E3) differed only in two nucleotides and one amino acid. The cloned SefA gene from S. enteritidis (E3) was submitted to the NCBI Genbank (EF553334).     

Characterization of beta-lactamases from urinary isolates of Escherichia coli in Tehran

BACKGROUND: Knowledge of antimicrobial resistance patterns in E. coli, the predominant pathogen associated with urinary tract infections (UTI) is important as a guide in selecting empirical antimicrobial therapy. METHODS: To describe the antimicrobial susceptibility of E. coli associated with UTI in a major university hospital in Tehran (Iran), seventy-six clinical isolates of E. coli were studied for susceptibility to beta-lactam antibiotics by the disc diffusion method and Minimal Inhibitory Concentrations determination. RESULTS: All isolates were resistant to ampicillin, amoxicillin and oxacillin. Resistance to the other tested antibiotics was shown to be 93.4% to cefradine, 76.3% to carbenicillin, 47.3% to cefazoline, 50% to cefalexin and 32.8% to cephalothin while 1.3% expressed resistance to cefoxitime, and 2.6% were resistant to ceftizoxime and ceftriaxone. Two isolates (2.4%) harbored extended spectrum beta-lactamases (ESBL) shown by the double disc diffusion method. Substrate hydrolysis by ultra violet spectroscopy showed that 87.4% harbored penicillinases, 9% produced cephlosporinases and 3.6% degraded both substrates. Clavulanic acid inhibited enzyme activity in 82.9%, of which 78.95% was penicillinases (group IIa) and 3.95% was cephalosporinases (group IIb) of the Bush classification system. The rest of the isolates (6.58 %) were placed in group IV beta-lactamases. No group III beta-lactamase was found, as EDTA inhibited none of the enzymes. DNA amplification by polymerase chain reaction using specific primers for ampC, TEM and SHV type beta-lactamases for all of the isolates showed that 47 organisms (60%) carried the TEM gene and 18 isolates (24%) harbored blaTEM and ampC genes. About 26% of the organisms harbored SHV type enzymes. CONCLUSION: These results indicate that E. coli can posses a variety of beta-lactamases that are responsible for beta-lactam resistance.     

IgM型单克隆抗体之scFv库的建立与筛选

从一个抗脂磷酸聚糖IgM型单克隆抗体细胞的mRNA中经RT—PCR克隆重链可变区（VH）和轻链可变区（VL）,然后将VH、Linker和VL连接成seFv,并克隆到载体pCANTAB-5E,转Escherichia coli TG1感受态细胞,构建了单链抗体库。通过辅助噬菌体M13KO7感染后,使单链抗体展示在噬菌体衣壳蛋白pⅢ的N端,得到噬菌体展示的抗体库。将抗体库用于“淘洗-富集-扩增”3轮以后,得到针对脂磷酸聚糖特异性的噬菌体抗体。测序结果表明,该scFv的VH基因序列全长390bp,编码127个氨基酸;VL基因序列全长341bp,编码113个氨基酸。二者均符合小鼠免疫球蛋白可变区基因特征,含有4个框架区（FR）、3个抗原互补决定区（CDR）及抗体特征性的2个半胱氨酸残基。