Analysis of electrophoretically heterogeneous lipopolysaccharides of Escherichia coli by immunoblotting

Abstract The purified lipopolysaccharide (LPS) from each of four Escherichia coli serogroups which was separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by periodic acid-silver staining, exhibited a different distribution of bands. The same serogroup-specific banding patterns were evident on immunoblots developed with homologous but not heterologous O-antisera. Thus, the electrophoretic heterogeneity of LPS was confirmed and the multiple bands seen on SDS-PAGE further shown to contain O-serogroup-specific antigenic determinants.     

Effect of oligomannoside-type glycopeptides in diarrheal disease of rabbits induced by Escherichia coli strain RDEC-1

We examined the effects of a soluble receptor-like complex carbohydrate (an oligomannoside-type glycopeptide) on adherence of a type 1 fimbriated rabbit enteropathogen, Escherichia coli strain RDEC-1. Oligomannoside-type glycopeptide, but not a non-glycosylated peptide mixture used as control, inhibited adherence of type 1 fimbriated RDEC-1 to both rabbit ileal brush borders and guinea pig erythrocytes in vitro. In contrast, during RDEC-1 infection of rabbits the receptor-like oligomannoside did not affect the presence and severity of diarrhea, fecal shedding of organisms, luminal colonization by RDEC-1, or enteroadherence of organisms.     

Cerebral inflammation contributes to encephalopathy and brain edema in acute liver failure: protective effect of minocycline

Encephalopathy and brain edema are serious complications of acute liver failure (ALF). The precise pathophysiologic mechanisms responsible have not been fully elucidated but it has been recently proposed that microglia‐derived proinflammatory cytokines are involved. In the present study we evaluated the role of microglial activation and the protective effect of the anti‐inflammatory drug minocycline in the pathogenesis of hepatic encephalopathy and brain edema in rats with ALF resulting from hepatic devascularisation. ALF rats were killed 6 h after hepatic artery ligation before the onset of neurological symptoms and at coma stages of encephalopathy along with their appropriate sham‐operated controls and in parallel with minocycline‐treated ALF rats. Increased OX‐42 and OX‐6 immunoreactivities confirming microglial activation were accompanied by increased expression of interleukins (IL‐1β, IL‐6) and tumor necrosis factor‐alpha (TNF‐α) in the frontal cortex at coma stage of encephalopathy in ALF rats compared with sham‐operated controls. Minocycline treatment prevented both microglial activation as well as the up‐regulation of IL‐1β, ΙL‐6 and TNF‐α mRNA and protein expression with a concomitant attenuation of the progression of encephalopathy and brain edema. These results offer the first direct evidence for central proinflammatory mechanisms in the pathogenesis of brain edema and its complications in ALF and suggest that anti‐inflammatory agents may be beneficial in these patients.     

Protective effect of C-reactive protein against the lethality induced by Vibrio vulnificus lipopolysaccharide

Vibrio vulnificus infection has attracted special interest because of its high mortality. A strong clinical association exists between hepatic dysfunction and increased morbidity and mortality from V. vulnificus infection. In this study, the effect of C-reactive protein (CRP), a typical hepatogenic acute phase protein, on the lethality induced by V. vulnificus lipopolysaccharide (LPS) was investigated in galactosamine-sensitized mice. The pretreatment of CRP, in a dose of at least 2 mg/kg, 2 hr before the challenge of LPS completely protected mice against the lethality by V. vulnificus LPS. The elevation of serum tumor necrosis factor-alpha (TNF-alpha) induced by LPS administration was not affected by CRP pretreatment. However, the LPS- or TNF-alpha-induced hepatotoxicity was completely prevented by CRP. These results indicate that CRP does not prevent the synthesis, but prevents the hepatotoxic action of TNF-alpha. The possibility that impaired production of acute phase proteins in patients with pre-existing hepatic dysfunction may predispose the higher risk of V. vulnificus infection needs to be evaluated further.     

Eicosanoids mediate nodulation reactions to bacterial Escherichia coli K 12 infections in larvae of the oriental blowfly, Chrysomya megacephala

Abstract  Nodulation is the predominant cellular defense reaction to bacterial challenges in insects. In this study, third instar larvae of Chrysomya megacephala were injected with bacteria, Escherichia coli K 12 (10⁶ CFU/mL, 2 μL), immediately prior to injection of inhibitors of eicosanoid biosynthesis, which sharply reduced nodulation response. Test larvae were treated with specific inhibitors of phospholipase A₂ (dexamethasone), cyclo-oxygenase (indomethacin, ibuprofen and piroxicam), dual cyclo-oxygenase/lipoxygenase (phenidone) and lipoxygenase (esculetin) and these reduced nodulation except esculetin. The influence of bacteria was obvious within 2 h of injection (5 nodules/larva), and increased to a maximum after 8 h (with 15 nodules/larva), and then significantly reduced over 24 h (9 nodules/larva). The inhibitory influence of dexamethasone was apparent within 2 h of injection (4 vs. 5 nodules/larva), and nodulation was significantly reduced, compared to control, over 24 h (5 vs. 8 nodules/larva). Increased dosages of ibuprofen, indomethacin, piroxicam and phenidone led to decreased numbers of nodules. Nodules continued to exist during the pupal stage. However, the effects of dexamethasone were reversed by treating bacteria-injected insects with an eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid. These findings approved our view that eicosanoid can mediate cellular defense mechanisms in response to bacterial infections in another Dipteran insect C. megacephala .     

Inhibitory effect of bacterial extracts on the activity in vitro of eukaryotic histone kinase

Abstract The in vitro ATP-dependent phosphorylation of histones catalyzed by bovine histone kinase is severely impaired by Escherichia coli cellular extracts. This effect is not due to a phosphoprotein phosphatase, or an ATPase, or a protease activity present in bacterial preparations. It seems rather attributable to a protein kinase inhibitor.     

The effect of the native bacterial community structure on the predictability of E. coli O157:H7 survival in manure‐amended soil

Aims: The survival capability of pathogens like Escherichia coli O157:H7 in manure‐amended soil is considered to be an important factor for the likelihood of crop contamination. The aim of this study was to reveal the effects of the diversity and composition of soil bacterial community structure on the survival time (ttd) and stability (irregularity, defined as the intensity of irregular dynamic changes in a population over time) of an introduced E. coli O157:H7 gfp‐strain were investigated for 36 different soils by means of bacterial PCR‐DGGE fingerprints. Methods and Results: Bacterial PCR‐DGGE fingerprints made with DNA extracts from the different soils using bacterial 16S‐rRNA‐gene‐based primers were grouped by cluster analysis into two clusters consisting of six and 29 soils and one single soil at a cross‐correlation level of 16% among samples per cluster. Average irregularity values for E. coli O157:H7 survival in the same soils differed significantly between clusters (P = 0·05), whereas no significant difference was found for the corresponding average ttd values (P = 0·20). The irregularity was higher for cluster 1, which consisted primarily of soils that had received liquid manure and artificial fertilizer and had a significant higher bacterial diversity and evenness values (P < 0·001). Conclusions: Bacterial PCR‐DGGE fingerprints of 36 manure‐amended soils revealed two clusters which differed significantly in the stability (irregularity) of E. coli O157 decline. The cluster with the higher irregularity was characterized by higher bacterial diversity and evenness. Significance and Impact of the Study: The consequence of a high temporal irregularity is a lower accuracy of predictions of population behaviour, which results in higher levels of uncertainty associated with the estimates of model parameters when modelling the behaviour of E. coli O157:H7 in the framework of risk assessments. Soil community structure parameters like species diversity and evenness can be indicative for the reliability of predictive models describing the fate of pathogens in (agricultural) soil ecosystems.     

Comparative assessment of the bactericidal effect of nanoparticles of copper oxide,silver, and chitosan-silver against Escherichia coli infection in broilers

Escherichia coli infection is considered one of the most economically important multi-systemic diseases in poultry farms. Several nanoparticles such as silver, chitosan, and copper oxide are known to be highly toxic to several microbes. However, there are no data concerning their success against in vivo experimental E. coli infection in broilers. Therefore, the present study was designed to investigate the bactericidal effect of low doses of CuO-NPs (5 mg/kg bwt), Ag-NPs (0.5 mg/kg bwt), and Ch-Ag NPs (0.5 mg/kg bwt) against E. coli experimental infection in broilers. One hundred chicks were divided into five groups as follows: (1) control; (2) E. coli (4 × 10⁸ CFU/ml) challenged; (3) E. coli +CuO-NPs; (4) E. coli +Ag-NPs; (5) E. coli +Ch-Ag NPs. The challenged untreated group, not NPs treated groups, recorded the lowest weight gain as well as the highest bacterial count and lesion score in all examined organs. The highest liver content of silver was observed in Ag-NPs treated group compared with the Ch-Ag NPs treated group. Our results concluded that Ch-Ag NPs not only had the best antibacterial effects but also acted as a growth promoter in broilers without leaving any residues in edible organs. We recommend using Ch-Ag NPs in broiler farms instead of antibiotics or probiotics.     

Optimization of a two-stage recombinant fermentation process: the dilution rate effect

The effects of dilution rates on the performance of a two-stage fermentation system for a recombinant Escherichia coli culture were studied. Dilution rate determines the apparent or averaged specific growth rate of a heterogeneous population of cells in the recombinant culture. The specific growht rate affects the genetic parameters involved in product formation in the second stage, such as plasmid stability, plasmid content, and specific gene expression rate. Kinetic models and correlations were developed for these parameters based on experimental data. Simulations of plasmid stability in the first stage showed that for longer fermentation periods, plasmid stability is better at higher dilution rates. However, the plasmid content is lower at these dilution rates. The optimal apparent specific growth rate for maximum productivity in the second stage was determined using two methods: (1) direct search for a constant specific growth rate, and (2) dynamic optimization using the maximum principle for a time-dependent specific growth rate profile. The results of the calculations showed that the optimum constant apparent specific growth rate for maximum over-all productivity is 0.40 h(-1). This coincides with the optimal specific growht rate for maximum plasmid content in the expressed stage. A 3.5% increase in overall productivity can be obtained by using a linear time dependent apparent specific growth rate control, mu(2)(t) = 0.0007t, in the course of the fermentation time.     

Role of Escherichia coli DnaK and DnaJ chaperones in spontaneous and induced mutagenesis and their effect on UmuC stability

The frequency of spontaneous as well as induced reversions of auxotrophic mutations in Escherichia coli AB1157 and its DeltadnaK and DeltadnaKdnaJ derivatives was estimated. The obtained results demonstrate that both mutants tested are characterized by elevated frequency of spontaneous reversions compared to their AB1157 parent. In contrast, the frequency of reversions induced by UV and MMS, i.e. agents inducing the SOS response, is reduced in DeltadnaJ and DeltadnaKdnaJ mutants, pointing to the possible defect of these mutants in error prone repair. Due to the fact that UmuC protein is one of the main players executing the error prone repair, its stability in DeltadnaJ and DeltadnaKdnaJ mutants was also studied. Reduced UmuC stability was demonstrated only in the DeltadnaKdnaJ mutant.     

Comparative substrate recognition by bacterial and fungal purine transporters of the NAT/NCS2 family

We compared the interactions of purines and purine analogues with representative fungal and bacterial members of the widespread Nucleobase-Ascorbate Transporter (NAT) family. These are: UapA, a well-studied xanthine-uric acid transporter of A. nidulans, Xut1, a novel transporter from C. albicans, described for the first time in this work, and YgfO, a recently characterized xanthine transporter from E. coli. Using transport inhibition experiments with 64 different purines and purine-related analogues, we describe a kinetic approach to build models on how NAT proteins interact with their substrates. UapA, Xut1 and YgfO appear to bind several substrates via interactions with both the pyrimidine and imidazol rings. Fungal homologues interact with the pyrimidine ring of xanthine and xanthine analogues via H-bonds, principally with N1-H and =O6, and to a lower extent with =O2. The E. coli homologue interacts principally with N3-H and =O2, and less strongly with N1-H and =O6. The basic interaction with the imidazol ring appears to be via a H-bond with N9. Interestingly, while all three homologues recognize xanthines with similar high affinities, interaction with uric acid or/and oxypurinol is transporter-specific. UapA recognizes uric acid with high affinity, principally via three H-bonds with =O2, =O6 and =O8. Xut1 has a 13-fold reduced affinity for uric acid, based on a different set of interactions involving =O8, and probably H atoms from positions N1, N3, N7 or N9. YgfO does not recognize uric acid at all. Both Xut1 and UapA recognize oxypurinol, but use different interactions reflected in a nearly 26-fold difference in their affinities for this drug, while YgfO interacts with this analogue very inefficiently.     

乳酸杆菌对大鼠细菌性阴道感染动物模型疗效观察

目的探讨乳酸杆菌对大鼠阴道菌群抑制及阴道菌群的调理作用。方法通过大鼠阴道混合感染葡萄球菌和大肠埃希菌的动物模型,以阴道分泌物的菌群分析和阴道黏膜炎症的治愈程度为指标,观察乳酸杆菌治疗组的治疗效果,用乳酸杆菌菌液冲洗治疗来去除大肠埃希菌和葡萄球菌在大鼠阴道的定植。结果通过各组大鼠阴道冲洗液检出的平均对数值比较,乳酸杆菌治疗组的大鼠阴道内大肠埃希菌和葡萄球菌的数量明显较对照组的数量减少（P〈0．05）,而且治疗组黏膜炎症的治愈程度显著好于对照组。结论乳酸杆菌能有效地去除大肠埃希菌和葡萄球菌的大鼠阴道定植和改善大鼠外阴的炎症症状。     

RAPID detection and quantification of E. coli O157/O26/O111 in minced beef by real-time PCR

AIM: To develop a real-time PCR detection procedure for Escherichia coli O111, O26 and O157 from minced meat. METHODS AND RESULTS: Strains (n = 8) of each of E. coli O26, E. coli O111 and E. coli O157 were inoculated at ca 10-20 CFU g(-1) into minced retail meat and enriched for 6 h at 41.5 degrees C as follows: E. coli O26 in tryptone soya broth (TSB) supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)); E. coli O111 in TSB supplemented with cefixime (50 microg l(-1)) and vancomycin (40 mg l(-1)); E. coli O157 in E. coli broth supplemented with novobiocin (20 mg l(-1)). DNA was extracted from the enriched cultures, and detected and quantified by real-time PCR using verotoxin (vt1 and vt2) and serogroup (O157 per gene; O26 fliC-fliA genes and O111 wzy gene) specific primers. CONCLUSIONS: The methods outlined were found to be sensitive and specific for the routine detection of E. coli O111, O26 and O157 in minced beef. SIGNIFICANCE AND IMPACT OF THE STUDY: The enrichment, isolation and detection procedures used in this study provide a rapid routine-based molecular method for the detection and differentiation of E. coli O26, O111 and O157 from minced meat.     

The regulation of porin expression in Escherichia coli: effect of turgor stress

The OmpF and OmpC porins are major outer membrane proteins of Escherichia coli. Their expression is affected by medium osmolarity such that OmpF is normally produced at low osmolarity and OmpC at high osmolarity. Potassium ion accumulation is a major means by which cells maintain their internal osmolarity in high osmolarity medium in the absence of organic osmolytes such as glycine-betaine. Starvation for potassium causes cells to become turgor stressed. The effect of turgor stress and potassium ion concentration on OmpF and OmpC expression was examined. It was found that ompF gene expression was switched off by turgor stress but there was no concomitant increase in OmpC. Instead, ompC expression responded to the accumulation of potassium ions by the cell in high osmolarity medium.     

老年大肠埃希菌血流感染患者的临床分析

为探讨老年大肠埃希菌血流感染患者的临床特点及药敏结果,回顾性分析徐州医科大学附属医院2013年1月-2016年12月340例大肠埃希菌血流感染老年患者的临床资料及药敏结果,采用SPSS软件进行分析。结果显示,患者均合并基础疾病(恶性肿瘤、高血压病、糖尿病、肺部感染等);感染途径主要为消化道(38.5%)、呼吸道(36.8%)和泌尿道(22.6%);检出产超广谱β-内酰胺酶(extended-spectrum β-lactamase,ESBL)大肠埃希菌96例(28.2%);ESBL阳性菌对氨苄西林、复方磺胺甲恶唑、环丙沙星、哌拉西林、第3代头孢、左氧氟沙星的耐药率均高;所有菌株均对碳青霉烯类敏感。结果提示,该院老年大肠埃希菌血流感染患者均合并基础疾病,常见感染途径是消化道、呼吸道和泌尿道,ESBL阳性大肠埃希菌除对碳青霉烯类抗菌药物敏感外,对多种抗菌药物耐药。     

Assessing immunocompetence in red palm weevil adult and immature stages in response to bacterial challenge and entomopathogenic nematode infection

Parasites and pathogens can follow different patterns of infection depending on the host developmental stage or sex. In fact, immune function is energetically costly for hosts and trade‐offs exist between immune defenses and life history traits as growth, development and reproduction and organisms should thus optimize immune defense through their life cycle according to their developmental stage. Identifying the most susceptible target and the most virulent pathogen is particularly important in the case of insect pests, in order to develop effective control strategies targeting the most vulnerable individuals with the most effective control agent. Here, we carried out laboratory tests to identify the most susceptible target of infection by infecting different stages of the red palm weevil Rhynchophorus ferrugineus (larvae, pupae, male, and female adults) with both a generic pathogen, antibiotic‐resistant Gram‐negative bacteria Escherichia coli XL1‐Blue, and two specific strains of entomopathogenic nematodes (EPNs), Steinernema carpocapsae ItS‐CAO1 and Heterorhabditis bacteriophora ItH‐LU1. By evaluating bacterial clearance, host mortality and parasite progeny release, we demonstrate that larvae are more resistant than adults to bacterial challenge and they release less EPNs progeny after infection despite a higher mortality compared to adults. Considering the two EPN strains, S. carpocapsae was more virulent than H. bacteriophora both in terms of host mortality and more abundant progeny released by hosts after death. The outcomes attained with unspecific and specific pathogens provide useful information for a more efficient and sustainable management of this invasive pest.     

Inhibition of growth rate of escherichia coli induced by extremely low-frequency weak magnetic fields

Cultures of Escherichia coli kept at 0 °C in a phosphate buffer solution were exposed to a sinusoidal weak 60- or 600-Hz magnetic field of strength 2 × 10^?3 Tesla. A decrease of more than 40% in bacterial count was observed after a 60-h exposure to the magnetic field. Electron micrographs of exposed bacteria show ruptured cell walls, possibly due to the breaking away of flagella under the influence of the sinusoidally varying electromotive force.     

Monitoring of active but non-culturable bacterial cells by flow cytometry

Flow cytometric signatures (i.e., light scatter, red and green fluorescence) were obtained for the active but non-culturable (ABNC) cells of E. coli and a coliform isolate H03N1, in seawater microcosms using BacLight, a live-dead assay kit from Molecular Probes (Eugene/Portland, OR). Previous studies have reported that there are two major adaptations, which cells undergo during the formation of ABNC states: cell wall toughening and DNA condensation. Therefore, we hypothesized that the matured ABNC forms should be more resistant to extreme temperature treatments (i.e., by freezing in liquid nitrogen and thawing at room temperature) than the normal and transition populations. It was shown that the membrane-compromised cells (comprising of normal wild-type and dead cells which are less resistant to rapid freeze thaw) could be differentiated from the matured ABNC using BacLight staining and fluorescence detection by flow cytometry. The population of ABNC cells, which could not be cultured using m-FC media (for the enumeration of fecal coliforms), was resuscitated in phosphate buffer saline followed by growth in Luria broth. Flow cytometry was thus able to detect and differentiate the ABNC cells against a mixed population comprising of culturable cells, transition populations, and dead cells. The results also showed that the formation of ABNC is as early as 2 days in seawater microcosms. By directly comparing the coliform levels enumerated by the BacLight based flow cytometry assays and m-FC technique, it was shown that the presence of coliforms can be undetected by the membrane filtration method.     

Differences in hydrogen exchange behavior between the oxidized and reduced forms of Escherichia coli thioredoxin.

Amide proton exchange of thioredoxin is used to monitor the structural effects of reduction of its single disulfide. An effective 3-5-proton difference between the oxidized and reduced protein form is observed early in proton out-exchange of the whole protein, which is independent of temperature in the range of 5-45 degrees C, indicating that redox-sensitive changes are probably not due to low-energy structural fluctuations. Medium resolution hydrogen exchange experiments have localized the redox-sensitive amide protons to two parts of the sequence that are distant from each other in the three-dimensional structure: the active-site turn and the first beta-strand. The sum of the proton differences observed in the peptides from these regions is equal to that of the whole protein, indicating that all redox-sensitive hydrogen exchange effects are observed in the peptide experiments. A model combining structural changes within the protein matrix with changes in the surface hydration properties is proposed as a mechanism for the communication between distant sites within the protein. Sound velocity and density measurements of reduced and oxidized thioredoxin are presented in the accompanying paper (Kaminsky, S.M. & Richards, F.M., 1992, Protein Sci. 1, 22-30).