Characteristics of β-Lactamase Synthesis in E. coli and K. pneumanie Strains in Nosocomial Infections

Background: Recently micro-organisms that synthesize extended-spectrum β-lactamase (ESBLs) were increased. The peculiarities of ESBL synthesis of Escherichia coli and Klebsiella pneumoniae strains that cause nosocomial urinary tract infections, surgical site infections and pneumonia in surgical clinic were studied. ESBL synthesis were observed 38.9% of E. coli strains obtained from urine, 92.3% of strains obtained from surgical site infections, and 50% of strains obtained from sputum. ESBL synthesis were observed 37.5% of K. pneumoniae strains obtained from urine, 85.7% of strains obtained from surgical site infections, and 60% of strains obtained from sputum. Different levels of ESBL synthesize of E. coli and K. pneumoniae strains isolated from different pattern is discussed. Conclusion. ESBL synthesis is common in E. coli and K. pneumoniae strains, which cause nosocomial infections. The frequency of occurrence of ESBL s synthesis among of these strains depends on clinical forms of nosocomial infections.

Evidence and Potential Antibacterial Mechanism of Chinese Traditional Medicine Compounds for the Development of E. coli

Astragalus membranaceus (huangqi), Allium sativum (garlic), Cinnamomum cassia (cinnamon), and Dolichos lablab L. (white hyacinth bean) are the traditional Chinese herbs that were used in prescriptions in treating diarrhea caused by bacterial infection. These herbs are relatively safe for use and investigation. This study aimed to investigate the effects of Astragalus membranaceus, Allium sativum, Cinnamomum cassia, and Dolichos lablab L. on the metabolism of Escherichia coli (E. coli). The growth rate of E. coli was monitored under the influence of each herb, revealing that Astragalus membranaceus and Allium sativum exhibited significant antibacterial activity, whereas Cinnamomum cassia and Dolichos lablab L. demonstrated moderate inhibitory effects on E. coli growth. Further inhibition zone testing allowed for the evaluation of each herb’s potency and the number of generations required for E. coli to develop resistance. Additionally, the impact of the four herbs on the expression of outer membrane protein A (OmpA) in E. coli was examined by using qPCR. The findings revealed that Astragalus membranaceus acted as a sustainable bactericide by inhibiting the growth and metabolism of E. coli MG1655 through the suppression of OmpA expression. These results suggest that Astragalus membranaceus has potential as a natural antimicrobial agent for treating E. coli infections.

Pharmacokinetics of Recombinant E. coli L-asparaginase in Rats

The distribution of ^(125)I recombinant E. coli L-asparaginase in tissues ororgans and the excretion in urine, feces and bile were studied with in vivo radioactive tracertechnique. The amount of radioactivity excreted in urine, feces and bile within 24 h afterintravenous administration of ^(125)I recombinant E. col L-asparaginase to rats was 68.95% ,4.44%and 5.36% of the dose respectively. ^(125)I recombinant E. coli L-asparaginase in plasma samples wasdetermined. The levels of structural intact molecule in plasma samples were evaluated by SDS-PAGEand bio-imaging analyzer system. Pharmacokinetic parameters were assessed with a model-dependentmethod. The concentration-time curves of recombinant E. coli L-asparaginase after intravenousinjection at 1 250 IU·kg^(-1), 2 500, IU·kg^(-1), 5 000 IU·kg^(-1) to rats were consistent withthe two-compartment model. The first and terminal elimination t_(1/2) were 0.52 ~ 0.63 h and 2.39 ~2.76 h respectively. AUC was linearly related to the doses. The results of distribution in tissuesor organs and excretion in urine suggested that the metabolites of the enzyme were cleared bymechanisms of urinary excretion. Pharmacokinetics parameters of recombinant E. coli L- asparaginasein rats are warranted for the design of future clinical trials.

Effects of Different Preservation Methods on Activity of Recombinant E. coli

[ Objective] To explore different preservation methods of recombinant E. coli and find out the optimal conditions for preservation. [ Method] The recombinant E. coli DH5cx transformed pcDNA.3 were respectively preserved at 4℃ and -70 ℃, and the activity was determined after dif- ferent time. [ Result] The number of living E. coll with high dilutions preserved at 4 ℃ was gradually increased within the first 7 d, peaked on Day 7, and then gradually decreased. The number of living E. coli, which were preserved in 8% glycerol at -70℃ when OD800 at 0.8, were significantly higher than that of other groups after different preservation time. [ Conclusion] The optimal storage time was 7 d for recombinant E. coli at 4 ℃. For preservation at -70 ℃, the bacteria should be in logarithmic growth phase and preserved in 8% glycerol.

Piglet Diarrhea and Technology Transfers to Prevent E. coli Infection at Small-Holder Pig Farms in Binh Dinh, Vietnam

The study aims at finding alternative interventions for the prevention ofE. coli infection in piglets so as to boost farmers＇ benefits and improve environment in the area. Data were collected from one experimental group consisting of 210 small pig holder farms and one control group of 70 farms. All of 280 litters and sows were analyzed and the techniques of early vaccination at 60-80 d of pregnant （time 1） were applied and repeated after 10 d with E. coli vaccine to produce antibodies through milk. The percentage of piglets with diarrhea caused by E. coli significantly decreased from 75.7% to 18.9%. Injection of E. coli vaccine for the pregnant sows created antibodies for young piglets through colostrums. The treatment of E. coli cost 45,420 Vietnam Dong （VND） per litter in the experimental group and that was 156,000 VND/litter in the control group. Use of betel-bokashi （bio-product as plant antibiotics and Lactobacillus） to treat piglet diarrhea was proved safe and effective. Litter size and average weight at birth and weaning were improved significantly （P 〈 0.05）. The average weaned weight of piglets and sows were 11.2 kg and 118.6 kg in the experimental group, compared to 9.4 kg and 89.9 kg in the control group, respectively. Piglets in the experimental group had good health, no diarrhea and a shorter weaning time （2 d） and mating interval 2.22 days earlier than those in the control group. The average profit of 210 households was significantly higher in the experiment group： feed-cost was much lower than that in the control group. Small-holder farms implementing the experiments of applying the techniques received more financial profit, which was 1,502,000 VND per year per sow, while those who did not apply the techniques gained only 223,000 VND per year per sow.

重组E. coli LLO/OVA刺激小鼠骨髓树突状细胞细胞因子表达的研究

目的 研究重组大肠杆菌疫苗E.coli LLO/OVA刺激小鼠骨髓树突状细胞的细胞因子表达,探讨该疫苗对BM-Dc的免疫刺激作用.方法 以E.coli OVA为对照,采用基因芯片、RT-PCR和ELISA在基因和蛋白质水平检测E.coli LLO/OVA刺激C57BL/6小鼠骨髓树突状细胞细胞因子表达情况.结果 经E.coli LLO/OVA刺激后4～24 h,BMDC出现一系列细胞因子基因表达上调,尤其在刺激后4～8 h BMDC的细胞因子基因上调表达明显,其中G-CSF和IFN-γ的表达明显高于E.coli OVA刺激后的BMDC RT-PCR检测也证实E.coli LLO/OVA刺激8 h后BMDC的IFN-γmRNA转录水平明显高于E.coli OVA刺激的BMDC FLISA结果显示E.coli LLO/OVA刺激BMDC后12～24 h,培养上清液内IFN-γ的含量明显高于E.coli OVA刺激后的BMDC,而IL-10的含量则明显降低.结论 E.coli LLO/OVA刺激小鼠BMDC上调表达一系列细胞因子,并且较E.coli OVA刺激的BMDC更明显上调G-CSF和IFN-γ基因表达.ILO作为重组疫苗E.coli OVA的免疫佐剂在刺激BMDC表达促进机体免疫的细胞因子中发挥重要作用.

Effect of Fragment Asp1961-Glu1978 in Fibronectin on the Expression of Triple-domain Polypeptide in E. coli

Two plasmids were constructed and used to express two triple-domain recombinant polypeptide of human fibronectin (FN). The cDNAs in plasmids code for two polypeptides, CH62 (Pro1239-Ser1515 of FN linked with Ala1690 -Val2049 through Met) and CH63 (CH62 without Ile1850-Glu1978). The expression level of CH62 in E. coli was very low, but that of CH63 was very high.The results suggests that Asp1961-Glu1978 in FN is a key sequence influencing the expression of triple-domain polypeptide in E. Coli. After being dissolved and renatured, CH63 can be purified by heparin-agarose affinity chromatography.Both of the cell-binding domains in the recombinant polypeptide were functional.The production of CH63 provides a fundamental basis for further study of recombinant products with better anti-metastasis function.

Establishment of an Infected Necrotizing Pancreatitis Model by Retrograde Pancreatic Duct Injection of Sodium Taurocholate and E. coli in Rats

A stable and reliable infected necrotizing pancreatitis （INP） model in rats was established in order to study the pathophysiological mechanism and pathological development role of INP and explore the new therapeutic methods for the diseases. Forty-six SD rats were randomly divided into 5 groups. The animals in group A received the injection of 5% sodium taurocholate into the pancreatic duct and those in group B underwent that of E. coli into the pancreatic duct. The rats in groups C, D and E were subjected to the injection of 5% sodium taurocholate in combination with different concentrations of E. coli （10^3, 10^4, 10^5/mE, respectively） into the pancreatic duct. The dose of injection was 0.1 mL/100 g and the velocity of injection was 0.2 mL/min in all the 5 groups. Eight h after the injection, the survival rate of animals was recorded and the surviving rats were killed to determine the serum content of amylase and perform pathological examination and germ cultivation of the pancreatic tissue. The results showed that acute necrotizing pancreatitis model was induced by injection of 5% sodium taurocholate into the pancreatic duct. The positive rate of germ cultivation in group A was 12.5%. The acute necrotizing pancreatitis model was not induced by injection of E. coli into the pancreatic duct and the positive rate of germ cultivation in group B was 0. The INP model was established in groups C to E. The positive rate of germ cultivation was 60%, 100% and 100% and 8-h survival-rate 100%, 100% and 70% in groups C, D and E, respectively. It was concluded that a stable and reliable model of INP was established by injection of 5% sodium taurocholate in combination with 10^4/mL E. coli into the pancreatic duct with a dose of 0.1 mL/100 g and a velocity of 0.2 mL/min. The pathogenesis of INP might be that the hemorrhage and necrosis of pancreatic tissue induced by sodium taurocholate results in weakness of pancreatic tissue in fighting against the germs. Meanwhile, the necrotic pancreatic tissue provides a good proliferative environment for the germs.

Isolation and Characterization of E. Coli O157 : H7 from Infected Newborn Calves in Northeast China

Escherichia coli O157 : H7 is a foodborne pathogen that poses a major threat to public health. Epidemiologic investigations have identified dairy cows, especially calves, are the principal reservoir of E. coli O157 : H7. In this study, based on the results, E. coli O157 : H7 was the main cause of E. coli disease outbreak in late October, 2015, and more than 90% of newborn calves died of serious diarrhea. Through further experiments, the drug sensitivity and resistance of the strain, the expression of the virulence gene and virulence pathogenicity were studied. E. coli O157 : H7 isolates were resistant to 12 antibiotics including penicillin, tetracycline and ampicillin, and were sensitive to eight antibiotics including cefoperazone, ceftazidime and amikacin. Resistance genes included tetB, strB, aadB, aphA, floR, TEM and virulence genes included stx1, eaeA and hlyA. Using specific pathogen free mice, the result showed that the isolate was pathogenic with a median lethal dose of 7.9×107 CFU · mL-1. This study described the pathogenesis and clinical manifestations of E. coli O157 : H7 infection. These results guided the use of antibiotics in prevent and control of bacterial infections in the future.

Identification of E. coli K12 chromosomal insertion sites of bacteriophage φ297

Objective:To identify the specific integration site of prophage φ297 in the host of E. coli K12 chromosome. Methods:Using molecular techniques such as Siebert PCR for walking from the int gene of prophage 297, which is similar to that of phage 933W to an unknown region in genomic DNA. A special adaptor is ligated to the ends of DNA fragments generated by digestion of genomic DNA with restriction enzymes that generates blunt ended fragments. Clone and subclone of PCR products, DNA sequencing and data analysis were used in this study. Results:The attL, attR and the core sequences were determined. The bacterial attachment site of phage φ297 was located in the yecE gene of E. coli K12. Conclusion:The phage φ297 integrates into the yecE gene of the E. coli K12 genome.

Expression of E. coli heat-labile enterotoxin B subunit in transgenic tobacco plants

Objective ： To construct plant transformation vector containing Escherichia coli heat-labile enterotoxin B subunit （LT-B） gene and generate LT-B transgenic tobacco plants. Methods： The LT-B coding sequence was amplified from pMMB68 by PCR, subcloned into middle vector pUCmT and binary vector pBI121 to obtain plant expression vector pBI-LTB, in which LT-B expression was controlled under the Cauliflower mosaic virus （CaMV） 35S promoter. The tobacco plants （Nicotiana tobacum L. Cuttivar Xanthi） were transformed by co-cultivating leaf discs method via Agrobacterium tumefaciens LBA4404 harboring the plant expression vector. The regenerated transgenic tobacco plants were selected by kanamycin and confirmed by PCR, Southern blot, Western blot and ELISA. Resuits： LT-B gene integrated in the tobacco genomic DNA and were expressed in 9 strains of transgenic tobacco plants. The yield was varied from 3. 36-10. 56 ng/mg total soluble tobacco leaf protein. Conclusion： The plant binary expression vector pBI-LTB was constructed successfully, and transgenic LT-B tobacco plants was generated, and confirmed by Southern blot. The protein LT-B expressed by engineered plants was identified by Western blot analysis and had the expected molecular weight of LT-B pentamer protein. This result is an important step close to developing an edible vaccine and supplying a mucasal immunoajuvant, which will contribute to the preven- tion of mucosaroute evading pathogen.

Effect of E. coli coli on Anti-disease Activities of Scailops: Argopecten irradians

The effect of acute E. coli challenge on the anti-disease activity of scallops Argopecten irra-dians is examined. The treatments of scallop from which hemolymph samples were taken for study included (1) control scallops, (2) sham-injected scallops, (3) PSW-injected scallops and (4) E. coli-injected scallops. From the beginning, the anti-disease activities of scallops are deter -mined at 12 hr and 24 hr.The concentrations of circulating hemocytes, the total serum protein concentrations and the activities of alkaline phosphatase, acid phosphatase and superoxide dismutase in the scallops Argopecten irradians are determined.Injection with E. coli results in a significant elevation in the concentration of circulating hemocytes and in the alkaline phosphatase activity and a significant decline in the total serum protein concentration and in the superoxide dismutase activity at 24 hr postchallenge. It shows that metabolism of bay scallop is expedited to adopt the challenge.

Effects of heavy metal on dielectric properties of E. coli revealed by dielectric spectroscopy

Dielectric spectroscopy of E. coli cell before and after exposure to heavy metals Cd^2＋ , Cu^2＋ , Zn^2＋ and Ca^2＋ was investigated. The results indicate that changes in dielectric spectra reflect effects of heavy metal on the structure and function of E. coli cells. Heavy metal can change membrane capacitance as well as pennittivity and conductivity of the cytoplasm. Changes in volume fraction suggested that dielectric measurement could monitor the growth of E. coli cells. These results demonstrated that dielectric spectroscopy was a potential effective technique for studying electric properties of biological cells.

Hepatitis A Virus PCR Analysis and <i>E. coli</i>Detection in Oysters at Oualidia Lagoon and Their Correlation

The present study aims to evaluate hepatitis A virus (HAV) prevalence and faecal contamination indicators Escherichia coli (E. coli) in oysters from Oualidia lagoon (Moroccan Atlantic coast) and to study the correlation between the two parameters. The survey was carried out on 87 samples of oysters (Crassostrea gigas) collected monthly between November 2015 and February 2017 from three sites corresponding to different oyster farms in the lagoon. Sanitary status of bivalve molluscs was assessed by E. coli enumeration using ISO 16649-3. Detection of hepatitis A virus, was carried out by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) according to ISO 15216-2 method. The prevalence of samples for which E. coli contamination exceeds the threshold of 230 E. coli/100g of flesh and intravalvular fluid (FIF) is 43%. HAV RNA was detected in 2% of the samples analyzed. This RNA was even detected in a sample meeting the bacterial criteria. Viral health surveillance of bivalve molluscs is therefore necessary before their delivery for human consumption.

Molecular Prevalence and Epidemiological Characteristics of Diarrheagenic <i>E. coli</i>in Children under 5 Years Old in the City of Koula-Moutou, East-Central Gabon

Background and Purpose: Diarrhoeagenic E. coli (DEC) is one of the germs responsible for childhood diarrhea in developing countries. This study aims at determining the prevalence of the five main pathotypes of DEC isolated from faeces of children under five years old with diarrhea or not, living in the city of Koula-Moutou. Methodology: Isolates of E. coli were phenotypically screened on chromIDTM agar and molecularly by multiplex PCR to detect the presence of enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorragic E. coli (EHEC) and enteroinvasive E. coli (EIEC). The evaluation of their sensitivity to 12 β-lactam antibiotic molecules was carried out by Kirby Bauer method. This method has also made it possible to characterize phenotypically the different β-lactamases produced. Results and Conclusion: Overall, at least one DEC pathovar was detected in the 63 E. coli strains with phenotypic and molecular frequencies of 63.5% and 68.5% respectively. Thus, ETEC (28.3%) and EHEC (28.3%) were the most frequent DEC in diarrheal isolates. ETEC/EHEC hybrid was recorded in both groups with rates of 7.5% in diarrheal cases and 10.0% for controls. The results showed produced carbapenemase type β-lactamases (31.7%), followed by ESBL (24.4%) and few produced high level penicillinases (4.9%). The DEC, in particular ETEC and EHEC are most likely the epidemiological agents responsible for childhood diarrhea in this study.

The Wheat Pathogenesis Related Protein (TdPR1.2) Ensures Contrasting Behaviors to <i>E. coli</i>Transformant Cells under Stress Conditions

The pathogenesis-related proteins 1 (PR-1) gene family play important roles in the plant metabolism in response to biotic and abiotic stresses. The wheat TdPR1.2 has been previously isolated and characterized. Here we showed by bio-informatic analysis that TdPR1.2 contains six cysteine residues that are conserved between all PR-1 proteins tested. Using ScanProsite tool, we found that TdPR1.2 structure has a CRISP family signature 1 and 2 located at the C-terminal part of the protein. Those two domains are conserved in many identified PR1.2 proteins in plants. Moreover, SignalIP-5.0 analysis revealed that TdPR1.2 contains a putative signal peptide formed by 25 amino acids at the N-terminal extremity. The presence of this signal peptide suggested that the mature proteins will be secreted after the cleavage of the signal sequence. Further, we investigate the role of the TdPR1.2 proteins in the growth of Escherichia coli transformants cells under different abiotic stresses. Our results showed that the full-length form of TdPR1.2 enhanced tolerance of E. coli against salt and osmotic stress but not to KCl. Moreover, TdPR1.2 protein confers bacterial tolerance to heavy metals in solid and liquid mediums. Based on these results, we suggest that the TdPR1.2 protein could play an important role in response to abiotic stress conditions.

EXPRESSION OF HBV PRE S1 PEPTIDE INE. Coli AND PRODUCT CHARACTERIZATION

HBV Pre S1 sequence is supposed to play an important role in the infection of HBV. Presence of Pre S1 /anti-Pre S1 in serum has valuble clinical imphations. In order to improve the study of Pre S1, Pre S1 sequence was overexpressed in E. coli as a fusion protein with MBP (Maltose- binding Protein), and anti-Pre S1 antiserum was elicited in rabbits by Pre S1 MBP purified by affinity chromatography. The recombinant plasmid constructed from PMAL-cRI expressed the 106aa Pre S1 sequence at the C terminal of MBP by tac promoter. The resulting Protein is about 54kD in size. Western-blot analysis confirmed its reactivity with antiserum derived from synthetic Pre S1 peptide and serum from patients with acute hepatitis B (AHB). ELISA showed that Pre S1-MBP and Dane Particles purified from AHB patient’s serum reacted with antiserum against synthetic Pre S1 peptlde,and this reaction was specifically inhibited by synthetic Pre S1 peptide. ELISA also demonstrated that antiserum against Pre S1-MBP reacted with synthetic Pre S1 peptide, but not with synthetic HCV peptide or HEV peptide.

提高重组质粒转化E. coli DH5α方法的研究

研究了质粒载体和外源DNA的浓度、酶切体系的酶切时间以及酶切产物的链接时间对转化效率的影响。结果表明,当pUC19质粒载体的浓度为25ng/μL,外源基因λDNA的浓度为100ng/μL,酶切时间3h,且连接时间为14h时,构建出的重组质粒转化大肠杆菌DH5α效率最高。

Microbiological Quality of Chicken Sold in Accra and Determination of D₁₀-Value of <i>E. coli</i>

Chicken is an excellent source of good quality protein, but it is highly susceptible to microbial contamination and often implicated in food borne disease. The microbiological quality of chicken at different retail outlets (supermarkets, local markets and farms) in Accra was investigated, and D10-values of E. coli in refrigerated and frozen retailed chicken was determined. The microbiological quality of chicken was studied by analyzing 27 chicken thigh samples collected from the retail outlets. D10-value of Escherichia coli was determined by using a linear regression model after gamma irradiation of inoculated chicken samples with doses of 0, 150, 300, 450, 600, 750 and 900 Gy. Mean total viable counts for the supermarkets, local markets and farms were 6.46, 6.91 and 6.57 log10 cfu/g respectively. Mean total coliform counts for the supermarkets, local markets and farms were 3.80, 3.46 and 3.14 log10 cfu/g respectively and the mean S. aureus counts were also 2.32, 2.28 and 2.70 log10 cfu/g respectively. There were no significant differences (p > 0.05) between the mean total viable count, total coliform counts and S. aureus count for the supermarkets, local markets and the farms. Mean counts of E. coli detected at the supermarket, local markets and farms were 1.27, 2.59 and 2.74 log10 cfu/g respectively. Salmonella spp. was detected in 2 out of the 27 samples. Fifty-two percent and 70% of samples respec-tively had total viable counts and total coliform counts within the microbial safety standards. Mean D10E. coli were 0.22 and 0.32 kGy in refrigerated and frozen chicken respectively. Presence of pathogenic bacteria in fresh chicken sold in some retail outlets in Accra was confirmed. Low D10-values of E. coli especially under refrigerated conditions suggest susceptibility to low dose irradiation and possibility of controlling spoilage and pathogenic microflora of fresh poultry.