A simple,robust enzymatic‐based high‐throughput screening method for antimicrobial peptides discovery against Escherichia coli

The indiscriminate usage of antibiotics has created a major problem in the form of antibiotic resistance. Even though new antimicrobial drug discovery programs have been in place from the last two decades, still we are unsuccessful in identifying novel molecules that have a potential to become new therapeutic agents for the treatment of microbial infections. A major problem in most screening studies is the requirement of high‐throughput techniques. Given this, we present here an enzyme‐based robust method for screening antimicrobial agent's active against Escherichia coli. This method is based upon the ability of the intracellular innate enzyme to cleave o‐nitrophenyl β‐d ‐galactopyranoside (non‐chromogenic) to o‐nitrophenolate (ONP) (chromogenic) upon the membrane damage or disruption. In comparison with the other currently available methods, we believe that our method provides an opportunity for real‐time monitoring of the antimicrobial agents action by measuring the ONP generation in a user‐friendly manner. Even though this method can be applied to other strain, our experience shows that one has to be careful especially when the pigments or metabolites present in the bacteria have the same wavelength absorbance. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Plasmid-associated bacteriocin production in a JK-type coryneform bacterium

Abstract The outer membrane of Escherichia coli K-12 has a variety of proteolytic activities. We were able to label several outer membrane proteins with [³H]diisopropylfluorophosphate (DFP). This suggests that they are serine proteases. The number of labelled proteins detected varied with the E. coli K-12 strain used. Strains bearing a tolC mutation, in addition, gave better labelling and/or had more labelled proteins. A previously described [³H]DFP-labelled outer membrane protein was shown not to be the TolC protein since it has a slightly lower M _r, it is not labelled more intensely in a TolC-overproducing strain, and it is still labelled in tolC mutant strains.     

F-specific RNA bacteriophages and sensitive host strains in faeces and wastewater of human and animal origin

Faeces of humans, pigs, cattle and chickens were investigated for the presence of somatic coliphages, F-specific bacteriophages and Escherichia coli strains sensitive to infection by F-specific phages. Attention was given to the possible effect of age and use of antibiotics on the prevalence of the FRNA phages and sensitive E . coli strains. Somatic coliphages were often detected in high numbers in all types of faeces. In contrast, FRNA phages were rarely detected in faeces from humans and cattle but more often in faeces from pigs and adult chickens. Samples from young chickens (with or without antibiotics) were consistently positive for FRNA phages (up to 3 × 10⁶ pfu/g). F-specific RNA phages were found in substantial numbers (> 10³ pfu/ml) in a variety of wastewaters: domestic, hospital, slaughterhouses and occasionally in 'grey water'. Their origin in wastewater was not clear. Strains from faeces usually belonged to serogroups I and IV. These types were also found in wastewater, as were group II and III strains. Serogroup II phages were abundant in wastewater of human origin but rare in faeces. Escherichia coli strains sensitive to infection by F-specific phages were common in faeces (overall 290/1081: 27%). No strains with fully derepressed F-pilus synthesis were detected among the sensitive strains. It is concluded that the occurrence of F-specific RNA bacteriophages in water points to sewage pollution rather than faecal pollution; the mechanism of replication of these organisms in wastewater is not understood.     

A methionine sulfoxide reductase in Escherichia coli that reduces the R enantiomer of methionine sulfoxide

It is known that Escherichia coli methionine mutants can grow on both enantiomers of methionine sulfoxide (met(o)), i.e., met-R-(o) or met-S-(o), indicating the presence of enzymes in E. coli that can reduce each of these enantiomers to methionine (met). Previous studies have identified two members of the methionine sulfoxide reductase (Msr) family of enzymes, MsrA and fSMsr, that could reduce free met-S-(o), but the reduction of free met-R-(o) to met has not been elucidated. One possible candidate is MsrB which is known to reduce met-R-(o) in proteins to met. However, free met-R-(o) is a very poor substrate for MsrB and the level of MsrB activity in E. coli extracts is very low. A new member of the Msr family (fRMsr) has been identified in E. coli extracts that reduces free met-R-(o) to met. Partial purification of FRMsr has been obtained using extracts from an MsrA/MsrB double mutant of E. coli.     

Overproduction of a Clostridium cellulolyticum endoglucanase by mutant strains of Escherichia coli

We have studied the expression of an endoglucanase from Clostridium cellulolyticum in mutant strains of Escherichia coli that overproduce haemolysin. When these mutants were transformed with plasmids encoding the endoglucanase, they showed a significantly enhanced endoglucanase activity, compared to transformed parental strains. Among the mutants, strain Hha-2 showed the highest production. We have identified the endoglucanase gene product synthesized in E. coli Hha-2/pBP8 and detected an increased amount of the enzyme parallel to the increase of endoglucanase activity. This was mainly localized in the periplasm and only a small percentage of it was found in the culture fluid.     

Detection of genetic markers of fecal indicator bacteria in Lake Michigan and determination of their relationship to Escherichia coli densities using standard microbiological methods

Lake Michigan surface waters impacted by fecal pollution were assessed to determine the occurrence of genetic markers for Bacteroides and Escherichia coli. Initial experiments with sewage treatment plant influent demonstrated that total Bacteroides spp. could be detected by PCR in a 25- to 125-fold-higher dilution series than E. coli and human-specific Bacteroides spp., which were both found in similar dilution ranges. The limit of detection for the human-specific genetic marker ranged from 0.2 CFU/100 ml to 82 CFU/100 ml culturable E. coli for four wastewater treatment plants in urban and rural areas. The spatial and temporal distributions of these markers were assessed following major rain events that introduced urban storm water, agricultural runoff, and sewage overflows into Lake Michigan. Bacteroides spp. were detected in all of these samples by PCR, including those with <1 CFU/100 ml E. coli. Human-specific Bacteroides spp. were detected as far as 2 km into Lake Michigan during sewage overflow events, with variable detection 1 to 9 days postoverflow, whereas the cow-specific Bacteroides spp. were detected in only highly contaminated samples near the river outflow. Lake Michigan beaches were also assessed throughout the summer season for the same markers. Bacteroides spp. were detected in all beach samples, including 28 of the 74 samples that did not exceed 235 CFU/100 ml of E. coli. Human-specific Bacteroides spp. were detected at three of the seven beaches; one of the sites demonstrating positive results was sampled during a reported sewage overflow, but E. coli levels were below 235 CFU/100 ml. This study demonstrates the usefulness of non-culture-based microbial-source tracking approaches and the prevalence of these genetic markers in the Great Lakes, including freshwater coastal beaches.     

大肠杆菌变种的两种分子生物学检测方法分析

目的:对大肠杆菌的一种重要的变种--肠出血性大肠杆菌O157-H7的几种检测方法进行比较研究.方法:以自动免疫磁珠收集系统(AIMS)、自动酶标免疫测试系统(VIDAS)与传统常规的分离方法进行对比分析.结果:运用自动免疫磁珠收集系统(AIMS)方法对80份可能含有肠出血性大肠杆菌O157-H7的实样进行检测,检出份数为6份,检出率为7.5％,而且在一周之内可以全部对上述检出实样进行鉴定.AIMS法能够检出浓度在10cfu/mL模拟实样之中的肠出血性大肠杆菌O157-H7,然后将此法与CHROMagar 0157琼脂板相结合,其效果则更为明显.而自动酶标免疫测试系统(VIDAS)与传统与的分离方法则检测的效果不佳,检出率为0.自动免疫磁珠收集系统(AIMS)检测方法与自动酶标免疫测试系统(VIDAS)、传统与的分离方法在检出率方面存在显著的统计学差异,P＜0.01.结论:运用自动免疫磁珠收集系统(AIMS)结合CHROMagar 0157琼脂板对出血性大肠杆菌O157-H7进行检测,检出率较高、灵敏度较高且快速便捷,可以用于O157-H7外环境检测与食品污染源的实际调查之中,应该对其加以广泛地推广并应用.     

Efficient transformation of Serratia marcescens with pBR322 plasmid DNA

Eight Serratia marcescens strains tested could be transformed with the plasmid pBR322. Transformants were selected on the basis of resistance to high levels of ampicillin (400 to 500 micrograms/ml). For six of the strains, the CaCl2- mediated transformation procedure developed for Escherichia coli was successful. For the other two strains, no transformants were obtained with the CaCl2-mediated transformation procedure unless the cells first received a heat treatment. Transformation frequency was dependent on DNA concentration, and no transformation was detected with linear pBR322 DNA. The stability and copy number of pBR322 were similar in S. marcescens and E. coli. As in E. coli, the pBR322 DNA was amplified in S. marcescens after inhibition of proteins synthesis. Based on these results, cloning in S. marcescens should be possible and pBR322 should be a useful cloning vehicle.     

New membrane-associated and soluble peptide methionine sulfoxide reductases in Escherichia coli

It is known that reactive oxygen species can oxidize methionine residues in proteins in a non-stereospecific manner, and cells have mechanisms to reverse this damage. MsrA and MsrB are members of the methionine sulfoxide family of enzymes that specifically reduce the S and R forms, respectively, of methionine sulfoxide in proteins. However, in Escherichia coli the level of MsrB activity is very low which suggested that there may be other enzymes capable of reducing the R epimer of methionine sulfoxide in proteins. Employing a msrA/B double mutant, a new peptide methionine sulfoxide reductase activity has been found associated with membrane vesicles from E. coli. Both the R and S forms of N-acetylmethionine sulfoxide, D-ala-met(o)-enkephalin and methionine sulfoxide, are reduced by this membrane associated activity. The reaction requires NADPH and may explain, in part, how the R form of methionine sulfoxide in proteins is reduced in E. coli. In addition, a new soluble Msr activity was also detected in the soluble extracts of the double mutant that specifically reduces the S epimer of met(o) in proteins.     

大肠杆菌产琥珀酸基因工程研究进展

生物合成琥珀酸摆脱了对不可再生战略资源石油的依赖,以其社会、经济和环境效益展现出良好的发展前景。野生型大肠杆菌的琥珀酸生产强度难以满足生物合成琥珀酸工业化的要求,但遗传背景清楚,容易改造。近年来,人们深入研究了大肠杆菌的琥珀酸代谢途径,通过强化大肠杆菌琥珀酸合成途径、抑制琥珀酸旁路代谢途径、构建产琥珀酸乙醛酸循环和有氧生产体系等多种基因工程策略,对大肠杆菌进行菌株改造和代谢进化筛选,提高了琥珀酸产量。综述了大肠杆菌产琥珀酸的基因工程研究进展。     

重庆地区儿童感染患者11039株大肠埃希菌的临床分布及耐药性

目的分析2010-2015年重庆地区儿童感染的11 039株大肠埃希菌的临床分布特征及耐药性,为合理应用抗菌药物和预防控制医院感染提供依据。方法分析大肠埃希菌对19种抗菌药物的耐药性,采用BD Phoenix 100 MIC法结合K-B纸片扩散法进行药敏试验,按美国临床实验室标准化委员会(CLSI)标准判断结果。结果大肠埃希菌总检出率为13.53%;大肠埃希菌在呼吸病房和新生儿病房的检出率高于其他病房;以痰标本多见,占62.22%(6869/11039);大肠埃希菌对氨苄青霉素耐药率最高(90.33%),对美罗培南、亚胺培南的耐药率低,分别为0.81%和0.78%。ESBLs平均检出率为50.98%(5628/11039)。结论大肠埃希菌的耐药性呈上升趋势,呼吸病房和新生儿病房是预防控制的重点科室。     

Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and beta-D-glucuronidase.

Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and beta-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E. coli, three major groups of diarrheagenic E. coli, three other non-coli Escherichia species, and various other gram-negative and -positive bacteria found in water. The genotypic assays were performed with hybridization probes generated by PCR amplification of 670- and 623-bp segments of the gadA/B (GAD) and uidA (GUD) genes, respectively. The GAD enzymes catalyze the alpha-decarboxylation of L-glutamic acid to yield gamma-aminobutyric acid and carbon dioxide, which are detected in the phenotypic assay by a pH-sensitive indicator dye. The phenotypic assay for GUD involves the transformation of 4-methylumbelliferyl-beta-D-glucuronide to the fluorogenic compound 4-methylumbelliferone. The GAD phenotypic assay detected the majority of the E. coli strains tested, whereas a number of these strains, including all representatives of the O157:H7 serotype and several nonpathogenic E. coli strains, gave negative results in the GUD assay. Both phenotypic assays detected some but not all strains from each of the four Shigella species. A strain of Citrobacter freundii was also detected by the GUD assay but not by the GAD assay. All E. coli and Shigella strains were detected with both the gadA/B and uidA probes. A few Escherichia fergusonii strains gave weak hybridization signals in response to both probes at 65 degrees C but not at 68 degrees C. None of the other bacterial species tested were detected by either probe. These results were consistent with previous reports which have indicated that the GAD phenotypic assay detects a wider range of E. coli strains than does the GUD assay and is also somewhat more specific for this species. The genotypic assays for the two enzymes were found to be equivalent in both of these respects and superior to both of the phenotypic assays in terms of the range of E. coli strains and isolates detected.     

Prevalence and molecular characterization of Escherichia coli O157:H7 on Irish lamb carcasses, fleece and in faeces samples

AIMS: To determine the prevalence, seasonal variation and virulence characteristics of Escherichia coli O157:H7 in lambs presented for slaughter in Ireland. METHODS AND RESULTS: Over a 13-month period, pre- and postchill carcass swabs, faeces and fleece samples from 1600 lambs were examined for the presence of E. coli O157:H7. Escherichia coli O157:H7 was isolated from 5.75% (23/400) of fleece samples, 1.5% (6/400) of pre- and 1% (4/400) of postchill carcass swabs but was not isolated in faeces (0/400). The present study detected no evidence of seasonal variation. Polymerase chain reaction analysis showed that both the vt1 and vt2 genes associated with clinical illness were carried by five of the E. coli O157:H7 isolates, while 24 of the remaining isolates carried the vt2 gene only. Phage typing detected four different subtypes: PT 32 (48.48%), PT 8 (12.12%), PT 31 (12.12%) and PT 21/28 (12.12%). CONCLUSIONS: Escherichia coli O157:H7 is present in lambs at slaughter in Irish abattoirs and the virulence profiles of these isolates reveals that they are potentially harmful to humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides crucial information indicating that sheep may be a significant contributing source to human E. coli O157:H7 infection.     

Survey of retail cheeses, dairy processing environments and raw milk for Escherichia coli O157 : H7

Escherichia coli was isolated from 58% (11/19) of retail soft and semi-soft cheeses tested, but E. coli O157 : H7 was not detected. The presence of E. coli in retail cheeses and the lack of baseline data on the prevalence of serotype O157 : H7 strains prompted us to survey ingredients and the environment in 15 cheese and dairy plants. Escherichia coli O157 : H7 was not detected in any of the 1104 samples tested, including 42 raw milk samples. These results suggest that serotype O157 : H7 is not prevalent within dairy product ingredients and processing environments.     

Genetic characterization of Bacillus subtilis odhA and odhB, encoding 2-oxoglutarate dehydrogenase and dihydrolipoamide transsuccinylase, respectively.

The 2-oxoglutarate dehydrogenase complex consists of three different subenzymes, the E1o (2-oxoglutarate dehydrogenase) component, the E2o (dihydrolipoyl transsuccinylase) component, and the E3 (dihydrolipoamide dehydrogenase) component. In Bacillus subtilis, the E1o and E2o subenzymes are encoded by odhA and odhB, respectively. A plasmid with a 6.8-kilobase-pair DNA fragment containing odhA and odhB was isolated. Functional E1o and E2o are expressed from this plasmid in Escherichia coli. Antisera generated against B. subtilis E1o and E2o expressed in E. coli reacted with antigens of the same size from B. subtilis. The nucleotide sequence of odhB and the terminal part of odhA was determined. The deduced primary sequence of B. subtilis E2o shows striking similarity to the corresponding E. coli protein, which made it possible to identify the lipoyl-binding lysine residue as well as catalytic histidine and aspartic acid residues. An mRNA of 4.5 kilobases hybridizing to both odhA and odhB probes was detected, indicating that odhA and odhB form an operon.     

Onopordopicrin from the new genus Shangwua as a novel thioredoxin reductase inhibitor to induce oxidative stress-mediated tumor cell apoptosis

Isolation and identification of natural products from plants is an essential approach for discovering drug candidates. Herein we report the characterization of three sesquiterpene lactones from a new genus Shangwua, e.g. onopordopicrin (ONP), C2, and C3, and evaluation of their pharmacological functions in interfering cellular redox signaling. Compared to C2 and C3, ONP shows the most potency in killing cancer cells. Further experiments demonstrate that ONP robustly inhibits thioredoxin reductase (TrxR), which leads to perturbation of cellular redox homeostasis with the favor of oxidative stress. Knockdown of the TrxR sensitizes cells to the ONP treatment while overexpression of the enzyme reduces the potency of ONP, underpinning the correlation of TrxR inhibition to the cytotoxicity of ONP. The discovery of ONP expands the library of the natural TrxR inhibitors, and the disclosure of the action mechanism of ONP provides a foundation for the further development of ONP as an anticancer agent.     

Factors influencing the presence and concentration of E. coli O157 and E. coli in farm waste on six cattle farms in North-West England

Aims:  To investigate the factors influencing the presence and burden of Escherichia coli O157 in farm wastes.
Methods and Results:  Wastes from six cattle farms were screened for the presence and concentration of E. coli O157 and E. coli on three occasions over a year and waste management data were collected. Sixty-three of 878 (7·1%) samples were positive for verocytotoxigenic Escherichia coli O157 and 664/875 (75·9%) for E. coli with detectable levels greater in fresh waste than in stored waste, pasture or dirty water.
Conclusions:  The turning/stirring of stored waste and the use of more than one store (allowing longer storage times) reduced the proportion of E. coli O157 positive samples. The presence of E. coli O157 significantly reduced from a high prevalence found in fresh faeces and stored waste to lower proportions in dirty water and pasture samples. Escherichia coli O157 was only detected on pasture when waste was spread from contaminated stores the day before sampling. A high prevalence of positive E. coli O157 samples were detected when cattle were re-housed.
Significance and Impact of the Study:  These findings help to support the importance of treating and storing farm waste, as well as providing evidence for the level of dilution of E. coli O157 from fresh waste to recently spread pastures.     

Clonal relationships among bovine pathogenic Escherichia coli producing surface antigen CS31A

Abstract Forty-two Escherichia coli strains producing surface antigen CS31A isolated from bovine infections were characterized with respect to OKH serotypes, outer membrane protein (OMP) elelctrophoretic patterns, allozymes for esterases A, B, C, I and biotypes. A large majority of the strains could be clustered in a limited number of groups of clonally related strains with diverse O serogroups. CS31A producing Escherichia coli strains thus appear to have a common genetic background and are representative of an important part of bovine pathogenic Escherichia coli .     

Detection of Escherichia coli cytotoxins by enzyme-linked immunosorbent assays

Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed to detect Escherichia coli cytotoxins. Wells were coated with monoclonal antibodies from hybridomas 13C4 and (or) 11E10, and biotin conjugates of these antibodies were used for detecting verotoxin 1 and Shiga-like toxin II, respectively. Sensitivities were about 100 and 200 cytotoxic doses, respectively. Verotoxin 2 was detected by ELISA with monoclonal antibody 11E10, but at a sensitivity of only about 4000 cytotoxic doses. ELISA results of polymyxin-treated cell extracts from cultures of 67 E. coli strains were in agreement with Vero cell assay as regards the presence and type of toxin.