Effect of colicin V on S. sonnei in vivo and in tissue culture.

The effect of the preparation of colicin V, freed from the endotoxin of the producer strain E. coli Ca-7 on Sh. sonnei was studied in the keratoconjunctival tests on guinea pigs and in the cell culture Hep-2. Colicin V was found to inhibit the development of dysenteric infection in vivo by delaying the incubation period. It has been demonstrated that the mentioned colicin blocks the process of adsorption of the causative agent on the surface of epithelial cells. Obviously, colicin V can also display bactericidal effect at the moment of intercellular migration of Shigellae, but it has no effect on bacteria which have penetrated into the cytoplasm of epithelial cells. The obtained data support the opinion of the role of colicins played in the development and course of infectious processes.     

Expression, processing, and assembly of foot-and-mouth disease virus capsid structures in heterologous systems: induction of a neutralizing antibody response in guinea pigs.

Plasmids containing the foot-and-mouth disease virus structural protein precursor (P1) and 3C protease genes or the P1 gene alone were expressed in Escherichia coli. A recombinant baculovirus containing the P1 gene was also generated and expressed in Spodoptera frugiperda cells. Expression of the P1 and 3C genes in E. coli resulted in efficient synthesis and processing of the structural protein precursor and assembly into 70S empty capsids. This material reacted with neutralizing monoclonal antibodies which recognize only conformational epitopes and elicited a significant neutralizing antibody response in vaccinated guinea pigs. Expression of the P1 gene in E. coli resulted in synthesis of an insoluble product, whereas in insect cells infected with the recombinant baculovirus a soluble product was synthesized. Both soluble and insoluble P1 reacted with a 12S-specific monoclonal antibody, but only soluble P1 elicited a neutralizing antibody response in guinea pigs.     

Detection and distribution of probiotic Escherichia coli Nissle 1917 clones in swine herds in Germany

AIMS: To verify the presence of Escherichia coli Nissle 1917 as a natural isolate in swine and to characterize in vitro probiotic properties as well as in vivo persistence in a feeding experiment. METHODS AND RESULTS: During studies on the intestinal microflora of pigs, we isolated E. coli Nissle 1917 sporadically from a pig population over a period of 1 year. The identity of the isolates as E. coli Nissle 1917 was verified by serotyping, Nissle-specific PCR, macrorestriction analysis (pulsed field gel electrophoresis) and the determination of in vitro probiotic properties in invasion and adhesion assays using a porcine intestinal epithelial cell line. Both the E. coli isolates and the E. coli Nissle 1917 strain showed strong reductions in adhesion of porcine enteropathogenic E. coli and invasion of Salmonella typhimurium with epithelial cells in vitro, with a probiotic effect. Screening of five epidemiologically unlinked swine farms and two wild boar groups showed one farm positive for E. coli Nissle 1917. A feeding experiment with four piglets showed viable E. coli Nissle 1917 in the intestine of three animals. CONCLUSIONS: The results of this study suggest that the E. coli Nissle 1917 strain is already partially established in swine herds, but the colonization of individual animals is variable. SIGNIFICANCE AND IMPACT OF THE STUDY: We report natural, long-term colonization and transmission of the probiotic E. coli Nissle 1917 strain in a swine herd, characterized individual persistence and colonization properties in swine and established an in vitro porcine intestinal epithelial cell model of probiotic action. The results of this study would have implications in the use of this strain as a probiotic in swine and contribute to a better understanding of the individual nature of intestinal bacterial persistence and establishment.     

Co-ordinate single-cell expression of LEE4- and LEE5-encoded proteins of Escherichia coli O157:H7

Escherichia coli O157:H7 is a zoonotic pathogen that can express a type III secretion system (TTSS) considered important for colonization and persistence in ruminants. E. coli O157:H7 strains have been shown to vary markedly in levels of protein secreted using the TTSS and this study has confirmed that a high secretion phenotype is more prevalent among isolates associated with human disease than isolates shed by healthy cattle. The variation in secretion levels is a consequence of heterogeneous expression, being dependent on the proportion of bacteria in a population that are actively engaged in protein secretion. This was demonstrated by indirect immunofluorescence and eGFP fusions that examined the expression of locus of enterocyte effacement (LEE)-encoded factors in individual bacteria. In liquid media, the expression of EspA, tir::egfp, intimin, but not map::egfp were co-ordinated in a subpopulation of bacteria. In contrast to E. coli O157:H7, expression of tir::egfp in EPEC E2348/69 was equivalent in all bacteria although the same fusion exhibited variable expression when transformed into an E. coli O157:H7 background. An E. coli O157:H7 strain deleted for the LEE demonstrated weak but variable expression of tir::egfp indicating that the elements controlling the heterogeneous expression lie outside the LEE. The research also demonstrated the rapid induction of tir::egfp and map::egfp on contact with bovine epithelial cells. This control in E. coli O157:H7 may be required to limit exposure of key surface antigens, EspA, Tir and intimin during colonization of cattle but allow their rapid production on contact with bovine gastrointestinal epithelium at the terminal rectum.     

Shiga-like-toxin-producing Escherichia coli in retail meats and cattle in Thailand.

Specific DNA probes were used to identify Shiga-like toxin I (SLT I)- and SLT II-producing Escherichia coli in vegetables, meats, cattle, and farm animals in Thailand. SLT-producing E. coli was isolated from 9% of market beef specimens, from 8 to 28% of fresh beef specimens at slaughterhouses, and from 11 to 84% of fecal specimens from cattle. Animals were frequently infected with several different SLT-producing E. coli types that hybridized with either the SLT I, SLT II, or both SLT probes. Of 119 SLT-producing E. coli isolates, 24% hybridized with the SLT I probe, 31% hybridized with the SLT II probe, and 44% hybridized with both SLT probes. The enterohemorrhagic E. coli plasmid probe hybridized with 64% (68 of 106) of SLT-producing E. coli isolates from food and cattle and with 8% (17 of 201) of E. coli isolates from pigs. No SLT-producing E. coli was detected in pigs. Seventy-six percent (26 of 34) of E. coli isolates that hybridized with the SLT II probe were cytotoxic to Vero but not to HeLa cells, suggesting that they produced the variant of SLT II. The high prevalence of SLT-producing E. coli in beef-producing animals suggests that exposure to animals and eating beef may pose a health risk for acquiring enterohemorrhagic E. coli infections in Thailand.     

Shiga-like-toxin-producing Escherichia coli in retail meats and cattle in Thailand

Specific DNA probes were used to identify Shiga-like toxin I (SLT I)- and SLT II-producing Escherichia coli in vegetables, meats, cattle, and farm animals in Thailand. SLT-producing E. coli was isolated from 9% of market beef specimens, from 8 to 28% of fresh beef specimens at slaughterhouses, and from 11 to 84% of fecal specimens from cattle. Animals were frequently infected with several different SLT-producing E. coli types that hybridized with either the SLT I, SLT II, or both SLT probes. Of 119 SLT-producing E. coli isolates, 24% hybridized with the SLT I probe, 31% hybridized with the SLT II probe, and 44% hybridized with both SLT probes. The enterohemorrhagic E. coli plasmid probe hybridized with 64% (68 of 106) of SLT-producing E. coli isolates from food and cattle and with 8% (17 of 201) of E. coli isolates from pigs. No SLT-producing E. coli was detected in pigs. Seventy-six percent (26 of 34) of E. coli isolates that hybridized with the SLT II probe were cytotoxic to Vero but not to HeLa cells, suggesting that they produced the variant of SLT II. The high prevalence of SLT-producing E. coli in beef-producing animals suggests that exposure to animals and eating beef may pose a health risk for acquiring enterohemorrhagic E. coli infections in Thailand.     

Antibody inhibition of HeLa cell invasion by Yersinia enterocolitica

Antisera were prepared in rabbits against formalized and heat-killed bacteria of Yersinia enterocolitica serotype O:5,27 and against formalized bacteria of serotype O:8. Both strains used for immunization demonstrated adhesion to and invasion of HeLa cells. Coating of the bacteria with antibody did not greatly alter adhesion (i.e., extracellular attachment) to HeLa cells; however, antibody against formalized bacteria of both serotypes inhibited HeLa cell invasion by the homologous and heterologous strains. The Fab fragments from purified immunoglobulins also demonstrated cross-reacting inhibition of HeLa cell invasion. Antibody against heat-killed bacteria of serotype O:5,27 had no inhibitory activity. Adsorption of the antiserum against formalized bacteria of serotype O:5,27 with lipopolysaccharide from the homologous strain removed anti-lipopolysaccharide antibody but did not remove the inhibitory activity. The antiserum against formalized bacteria of serotype O:8 showed no antibody against lipopolysaccharide from serotype O:5,27 and no agglutinins against heat-killed bacteria of this strain. From these results, it is tentatively suggested that protein structures are important in mediating epithelial cell invasion by Y. enterocolitica.     

Plasmid association and nucleotide sequence relationships of two genes encoding heat-stable enterotoxin production in Escherichia coli H-10407.

Plasmid DNA from enterotoxigenic Escherichia coli strains H-10407 and H-10407-P was examined for nucleotide sequence homology to two E. coli genes encoding infant mouse-active heat-stable enterotoxins (ST). A 62-megadalton plasmid of strain H-10407 contained sequences homologous to the gene encoding a toxin designated STIb, previously isolated from a human isolate of E. coli. A 42-megadalton plasmid of strains H-10407 and H-10407-P contained sequences homologous to the gene encoding a toxin designated STIa, previously isolated from bovine and porcine isolates of E. coli.     

Invasion of bovine epithelial cells by verocytotoxin-producing Escherichia coli O157:H7

The ability of verocytotoxin-producing Escherichia coli (VTEC) O157:H7 to enter selected human (RPMI-4788 and HeLa) and bovine (MAC-T, mammary secretory; MDBK, kidney) epithelial cell lines was evaluated. All VTEC evaluated efficiently entered RPMI-4788 and MAC-T cell lines. VTEC entered MDBK cells at approximately 4% of MAC-T cells. VTEC were not able to invade HeLa cells. Presence of plasmid had no influence on efficiency of entry, nor did production of shiga-like toxin (SLT I or SLT II). Internalization required microfilaments, but not microtubules. Two types of adherence, localized and diffuse, were exhibited depending on isolate and cell line evaluated. Ability of VTEC to invade bovine mammary epithelial cells may be important in pathogenesis in the bovine, may indicate a route by which raw milk may potentially become contaminated, and may provide a reservoir of bacteria for the contamination of workers, equipment and carcass at time of slaughter.     

Invasion of tissue culture cells by diarrhoeagenic strains of Escherichia coli which lack the enteroinvasive inv gene

Abstract Invasive Escherichia coli strains of certain serotypes invade by the same mechanism as the Shigella sp. It has been proposed that invasion of epithelial cells by EPEC strains may also occur; this is a previously overlooked property. In the present study E. coli strains isolated from patients with diarrhoea or ulcerative colitis, lacking the inv plasmid mediating classical invasion, but hybridizing with probes for different adhesins, were analyzed for their ability to invade HeLa and Caco-2 cells. The majority of strains invaded Caco-2 cells to a higher extent than HeLa cells. Adhesion to Caco-2 cells was a prerequisite for subsequent invasion of the cells but EAF, eae , EAgg and other known virulence factors were not sufficient to mediate invasion. In 8/9 E. coli strains invasion was enhanced after growth under iron restriction. Growth during anaerobic conditions did not influence subsequent invasion by E. coli strains whereas 6/9 strains had their invasive ability significantly decreased after growth in the presence of 1% glucose. The invasive process was inhibited by mannose but not by lactose, fucose or galactose. Our data indicate that strains of E. coli may invade Caco-2 cells by novel mechanisms which require adhesion to the cells but which differ from those of Salmonella sp., Yersinia sp., Shigella sp. and classical enteroinvasive E. coli .     

Bioluminescent high-throughput assay for the bacteria adherence to the tissue culture cells

The goal of this study was develop a rapid high-throughput method for the assessment of the bacterial adhesion to tissue culture cells and test this method by investigation of the adhesion and growth of pathogenic and non-pathogenic Escherichia coli strains in the presence of HeLa human epithelial cells. Fifteen strains of E. coli were transformed with a plasmid carrying the entire lux operon of Photorhabdus luminescens to make them bioluminescent. By using the Time-to-Detection approach and bioluminescence imaging in microplate format, the adherence and growth of bacteria in tissue culture medium in the presence of HeLa cells was monitored. It was observed that Eagle's minimal essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS) significantly inhibited growth of E. coli. However, in the presence of HeLa cells the detected growth of E. coli was similar to the growth observed in LB medium. It was established that the initial number of E. coli cells present in the microplate directly correlated with the time necessary for the bioluminescence signal to reach the threshold level, hence allowing the accurate assessment of the adhered cells within 8-10 h. Neither bacterial adherence nor growth kinetics correlated with the pathogenicity of the strain though they were strain-specific. The developed approach provided new information on the interaction of E. coli with epithelial cells and could be used for both pathogenicity research and for the screening of potential therapeutic agents for the ability to minimize pathogen colonization of human tissues.     

Growth kinetics of coliform bacteria under conditions relevant to drinking water distribution systems.

The growth of environmental and clinical coliform bacteria under conditions typical of drinking water distribution systems was examined. Four coliforms (Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes, and Enterobacter cloacae) were isolated from an operating drinking water system for study; an enterotoxigenic E. coli strain and clinical isolates of K. pneumoniae and E. coli were also used. All but one of the coliforms tested were capable of growth in unsupplemented mineral salts medium; the environmental isolates had greater specific growth rates than did the clinical isolates. This trend was maintained when the organisms were grown with low levels (less than 1 mg liter-1) of yeast extract. The environmental K. pneumoniae isolate had a greater yield, higher specific growth rates, and a lower Ks value than the other organisms. The environmental E. coli and the enterotoxigenic E. coli strains had comparable yield, growth rate, and Ks values to those of the environmental K. pneumoniae strain, and all three showed significantly more successful growth than the clinical isolates. The environmental coliforms also grew well at low temperatures on low concentrations of yeast extract. Unsupplemented distribution water from the collaborating utility supported the growth of the environmental isolates. Growth of the K. pneumoniae water isolate was stimulated by the addition of autoclaved biofilm but not by tubercle material. These findings indicate that growth of environmental coliforms is possible under the conditions found in operating municipal drinking water systems and that these bacteria could be used in tests to determine assimilable organic carbon in potable water.     

Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa sublines and recovery of a HeLa clonal line persistently infected with incomplete virus

Rustigian, Robert (Tufts University School of Medicine, Boston, Mass.). Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa clonal line persistently infected with incomplete virus. J. Bacteriol. 92:1805-1811. 1966.-The effect of viral antibody on persistent infection of HeLa cells by the Edmonston strain of measles virus was investigated by culturing cells from three persistently infected clones in medium supplemented with human immune globulin. The three infected HeLa clones were isolated from a persistently infected parent line. Two sublines which were grown in the presence of measles antibody developed a nonyielder state, wherein there is no detectable virus infectious for normal HeLa cultures. There is, however, continued synthesis of intracellular viral antigen and formation of viral intracytoplasmic inclusion bodies. The development of a nonyielder state was associated with a marked decrease in the degree of hemadsorption in cultures of both sublines. Further studies of the viral properties of non-yielder HeLa cell populations were made with a clone obtained from one of these sublines by plating under antibody. Persistent infection in this line was characterized by synthesis of incomplete virus even when the cells were cultured thereafter in anti-body-free medium. This was evidenced by (i) failure to recover infectious virus from the clonal population despite continued formation of intracellular viral antigen and viral intracytoplasmic inclusion bodies in a majority of the cells, (ii) the presence of only a few cells with surface viral antigen(s) including hemagglutinin, and (iii) the relatively weak antibody response to viral envelope antigen(s) after injection of cells into guinea pigs.     

Differential adhesion of major surface proteins 1a and 1b of the ehrlichial cattle pathogen Anaplasma marginale to bovine erythrocytes and tick cells

Anaplasma marginale is a tick-borne ehrlichial pathogen of cattle for which six major surface proteins (MSPs) have been described. The MSP1 complex, a heterodimer composed of MSP1a and MSP1b, was shown to induce a protective immune response in cattle and both proteins have been identified as putative adhesins for bovine erythrocytes. In this study the role of MSP1a and MSP1b as adhesins for bovine erythrocytes and tick cells was defined. msp1alpha and msp1beta1 genes from the Oklahoma isolate of A. marginale were cloned and expressed in Escherichia coli K-12 under the control of endogenous and tac promoters for both low and high level protein expression. Expression of the recombinant polypeptides was confirmed and localised on the surface of transformed E. coli. The adhesion properties of MSP1a and MSP1b were determined by allowing recombinant E. coli expressing these surface polypetides to react with bovine erythrocytes, Dermacentor variabilis gut cells and cultured tick cells derived from embryonic Ixodes scapularis. Adhesion of the recombinant E. coli to the three cell types was determined using recovery adhesion and microtiter haemagglutination assays, and by light and electron microscopy. MSP1a was shown by all methods tested to be an adhesin for bovine erythrocytes and both native and cultured tick cells. In contrast, recombinant E. coli expressing MSP1b adhered only to bovine erythrocytes and not to tick cells. When low expression vectors were used, single E. coli expressing MSP1a was seen adhered to individual tick cells while reaction of tick cells with the E. coli/MSP1a/high expression vector resulted in adhesion of multiple bacteria per cell. With electron microscopy, fusion of E. coli cell membranes expressing MSP1a or MSP1b with erythrocyte membranes was observed, as well as fusion of tick cell membranes with E. coli membranes expressing MSP1a. These studies demonstrated differential adhesion for MSP1a and MSP1b for which MSP1a is an A. marginale adhesin for both bovine erythrocytes and tick cells while MSP1b is an adhesin only for bovine erythrocytes. The role of the MSP1 complex, therefore, appears to vary among vertebrate and invertebrate hosts.     

HeLa cell invasion by a strain of enteropathogenic Escherichia coli that lacks the O-antigenic polysaccharide

The interaction with HeLa cells of an enteropathogenic Escherichia coli (EPEC) strain and its plasmid-cured derivative strain was examined. An O111:NM EPEC strain B171 harbours a 54 megadalton plasmid (pYR111) necessary for the expression of both localized adherence (LA) to HeLa cells and the O-repeating side chain of the lipopolysaccharide. Under light microscopy, the plasmid-cured derivative strain B171-4 was observed to interact with HeLa cells in a pattern distinct from LA. Transmission electron microscopy showed that the bacteria were internalized by HeLa cells. In contrast, strain B171 induced pedestal-like projections and invaginations of the plasma membrane, but was never completely internalized. A quantitative assay to determine the number of internalized bacteria revealed that strain B171-4 was internalized at levels 30-70-fold higher than those of avirulent E. coli strains. Cytochalasin B reduced the levels of internalization of both strain B171-4 and an enteroinvasive E. coli strain (E11), but did not affect LA by strain B171. These results suggest that EPEC strain B171 may carry a specific chromosomally determined surface factor needed to initiate internalization by HeLa cells. However, a plasmid-determined factor alters the nature of this interaction; the combined effects of the chromosomal and plasmid determinants lead to the characteristic attachment of the bacteria in clusters on the surface of the eukaryotic cell.     

Growth kinetics of coliform bacteria under conditions relevant to drinking water distribution systems.

The growth of environmental and clinical coliform bacteria under conditions typical of drinking water distribution systems was examined. Four coliforms (Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes, and Enterobacter cloacae) were isolated from an operating drinking water system for study; an enterotoxigenic E. coli strain and clinical isolates of K. pneumoniae and E. coli were also used. All but one of the coliforms tested were capable of growth in unsupplemented mineral salts medium; the environmental isolates had greater specific growth rates than did the clinical isolates. This trend was maintained when the organisms were grown with low levels (less than 1 mg liter-1) of yeast extract. The environmental K. pneumoniae isolate had a greater yield, higher specific growth rates, and a lower Ks value than the other organisms. The environmental E. coli and the enterotoxigenic E. coli strains had comparable yield, growth rate, and Ks values to those of the environmental K. pneumoniae strain, and all three showed significantly more successful growth than the clinical isolates. The environmental coliforms also grew well at low temperatures on low concentrations of yeast extract. Unsupplemented distribution water from the collaborating utility supported the growth of the environmental isolates. Growth of the K. pneumoniae water isolate was stimulated by the addition of autoclaved biofilm but not by tubercle material. These findings indicate that growth of environmental coliforms is possible under the conditions found in operating municipal drinking water systems and that these bacteria could be used in tests to determine assimilable organic carbon in potable water.     

Properties of hog cholera viruses recently isolated in Japan

Hog cholera (HC) viruses newly isolated in Japan in 1980 and 1981 were examined for pathogenicity and serological properties by the neutralization test with antisera against bovine viral diarrhea-mucosal disease (BVD . MD) and HC viruses. Five of 23 isolates examined were neutralized poorly by BVD . MD antibody, but well by HC antibody. On the contrary, 15 isolates were neutralized readily and two isolates moderately by BVD . MD antibody. The other one reacted poorly with either HC or BVD . MD antibodies. The isolate neutralized poorly by BVD . MD antibody was more highly pathogenic than those neutralized readily. It was concluded that the antigenic properties and pathogenicity of the HC viruses were not monotype , and that HC viruses varying in antigenicity and pathogenicity were present in the field.     

大肠杆菌血脑屏障侵袭基因 ibeB的稳定转染 能够促进 HeLa 细胞片状伪足的形成

为进一步探讨大肠杆菌血脑屏障侵袭基因 ibeB 的生物学特性,将 ibeB 基因克隆至真核表达载体,采用稳定转染的方法,对 ibeB 基因在 HeLa 细胞中的表达所产生的影响进行了研究 . 结果发现：经 ibeB 基因稳定转染的 HeLa 细胞向外周伸展,细胞骨架的组分———微丝发生了重新排布,形成了明显的片状伪足,细胞周边形成较多的膜皱褶结构;细胞对底物的粘附能力和伸展能力显著增强;细胞的粘着斑标志性蛋白 Vinculin 表达增强 . 这些结果为研究片状伪足的形成机制提供了模型 .     

Antibiotic resistance in bacteria from magpies (Pica pica) and rabbits (Oryctolagus cuniculus) from west Wales

The prevalence of antibiotic-resistant bacteria in wild animal and bird populations is largely unknown, with little consistency among the few published reports. We therefore examined intestinal bacteria from magpies ( Pica pica ) and rabbits ( Oryctolagus cuniculus ) collected in rural west Wales. Escherichia coli isolates resistant to multiple antibiotics were grown from eight of 20 magpies trapped in spring, 1999 and one of 17 in spring, 2000; the most prevalent resistance trait among these isolates was to tetracycline, but resistances to ampicillin, chloramphenicol, kanamycin, sulphonamide, tetracycline and trimethoprim were also found. Tetracycline-resistant Enterococcus spp. were found in one of 20 magpies in 1999 and three of 17 in 2000. Only one resistant E. coli isolate was detected among gut bacteria from 13 rabbits, and this strain was resistant only to tetracycline. Differences in the prevalence of resistance between bacteria from rabbits and magpies may reflect differences in diet: rabbits graze field edges, whereas magpies are omnivorous and opportunistic. The resistance genes found in E. coli isolates from magpies mostly corresponded to those common among human isolates, but those conferring tetracycline resistance were unique.     

A highly conserved endonuclease activity present in Escherichia coli, bovine, and human cells recognizes oxidative DNA damage at sites of pyrimidines.

We have compared the sites of nucleotide incision on DNA damaged by oxidizing agents when cleavage is mediated by either Escherichia coli endonuclease III or an endonuclease present in bovine and human cells. E. coli endonuclease III, the bovine endonuclease isolated from calf thymus, and the human endonuclease partially purified from HeLa and CEM-C1 lymphoblastoid cells incised DNA damaged with osmium tetroxide, ionizing radiation, or high doses of UV light at sites of pyrimidines. For each damaging agent studied, regardless of whether the E. coli, bovine, or human endonuclease was used, the same sequence specificity of cleavage was observed. We detected this endonuclease activity in a variety of human fibroblasts derived from normal individuals as well as individuals with the DNA repair deficiency diseases ataxia telangiectasia and xeroderma pigmentosum. The highly conserved nature of such a DNA damage-specific endonuclease suggests that a common pathway exists in bacteria, humans, and other mammals for the reversal of certain types of oxidative DNA damage.