Relation Between Deoxyribonucleic Acid and Intracytoplasmic Membranes in Escherichia coli O111a1

The possibility of a relationship between intracytoplasmic membranes and deoxyribonucleic acid (DNA) in Escherichia coli O111a(1) has been investigated. To facilitate this investigation, a simple enzymatic assay for the amount of internal membrane present in a culture was developed. This assay was then used to show that the appearance of intracytoplasmic membranes is accompanied by an increase in the DNA content of the cells. Electron micrographs have confirmed this observation and have shown DNA to be in contact with the intracytoplasmic membranes. Extensive membranes were observed at sites of apparently unsuccessful attempts at cell division. These observations led to the conclusion that the internal membrane formed by strain O111a(1) represents "extra" membrane, which is functional in that it contains sites for DNA replication, but is produced in excess because the organism is somehow defective in its regulation of membrane synthesis.     

Formation and Ultrastructure of Extra Membranes in Escherichia coli

A temperature-sensitive strain of Escherichia coli (strain 0111a(1)) was shown to accumulate membranous structures at 40 C. These "extra membranes" appeared as vesicles or whorls (or both), depending on the time of growth at 40 C. After 2 hr of growth at 40 C, only vesicles were observed in E. coli 0111a(1) cells; both vesicles and whorls were apparent after 6 hr. The number of cells which contained both types of extra membrane reached a maximum value (75%) after 10 hr of growth at 40 C. Extra membrane production was also studied by using temperature shifts. In shift-up experiments, cells grown at 30 C into early stationary phase accumulated extra membrane after a shift to 40 C. The percentage of E. coli 0111a(1) cells containing extra membrane decreased significantly after a shift from 40 to 30 C. Phase- and electron-microscopic observations indicated that E. coli 0111a(1) cells grown at 40 C were larger than E. coli 0111: B(4) cells grown at either temperature. The ratio of optical density per cell and cell measurements obtained from quantitative electron microscopy confirmed that E. coli 0111a(1) cells grown at 40 C were about twice as large. Microdensitometer traces indicated that the dimension of a single membrane of either whorls or vesicles was 5.4 nm in peak-to-peak distance (8.8 nm total thickness).     

Determination of Glycosyltransferase Specificities for the Escherichia coli O111 O Antigen by a Generic Approach

We describe a bacterial strain developed to facilitate the determination of glycosyltransferase (GT) specificities for O antigens of known structure and gene cluster sequence. For proof of principle for the approach, the strain was used to determine the specificity of the Escherichia coli O111 O-antigen GT genes.     

Molecular serotyping of Escherichia coli O111:H8

Accurate Escherichia coli serotyping is critical for pathogen diagnosis and surveillance of non-O157 Shiga-toxigenic strains, however few laboratories have this capacity. The molecular serotyping protocol described in this paper targets the somatic and flagellar antigens of E. coli O111:H8 used in traditional serotyping, and can be performed routinely in the laboratory.     

Immunomagnetic separation of Escherichia coli O26, O111 and O157 from vegetables

AIMS: Raw fruits and vegetables have been increasingly associated with human infections caused by Shiga toxin-producing Escherichia coli. This study evaluates the isolation and detection of E. coli O26, O111 and O157 from vegetable samples using immunomagnetic particles. METHODS AND RESULTS: Standard cultivation and immunomagnetic separation (IMS) procedures were compared. It was found that immunomagnetic particles could efficiently concentrate E. coli cells, detecting significantly more bacteria than with standard cultivation procedures. CONCLUSION: Bacteria were detected in 93-100% of the inoculated samples using the IMS procedure, but only 36-93% samples tested by standard cultivation procedures were found to be positive. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that E. coli O26, O111 and O157 immunomagnetic particles can be a very useful and efficient tool for the detection of E. coli strains in raw vegetables, and could probably be used with samples of animal origin.     

Polymyxin-based enzyme-linked immunosorbent assay for the detection of Escherichia coli O111 and O26

Polymyxin-based enzyme-linked immunosorbent assay (polymyxin-ELISA) systems were developed for the detection of Escherichia coli O111 and O26 in ground beef after enrichment. Polymyxin immobilized in the wells of a microtiter plate served as a high affinity adsorbent for lipopolysaccharide (LPS) antigens, which were detected immunoenzymatically using commercially available anti-E. coli O111 or anti-E. coli O26 antisera. The polymyxin-ELISA sensitively detected E. coli strains bearing the O111 and O26 LPS antigens, discriminating between these target strains and a panel of various non-target Gram negative and Gram positive bacteria. The detection of E. coli O111 and O26 strains inoculated into ground beef was achieved after enrichment in either modified trypticase soy broth (TSB) with novobiocin, or the serotype-specific medium TSB supplemented with cefixime and vancomycin (E. coli O111), and the same medium containing potassium tellurite (E. coli O26). The polymyxin-ELISA shows promise as a rapid, simple and inexpensive screening tool for E. coli O111 and O26 in enrichment cultures of ground beef.     

Murine Monoclonal Antibodies Specific for Lipopolysaccharide of Escherichia coli O26 and O111

Monoclonal antibody (MAb) 12F5 reacted with 35 Escherichia coli O26 isolates and cross-reacted with 1 of 365 non-E. coli O26 isolates. MAb 15C4 reacted with 30 E. coli O111 strains and 8 Salmonella O35 strains (possessing identical O antigen) but not with 362 other bacterial strains. Lipopolysaccharide immunoblots confirmed MAb O-antigen specificity.     

Murine monoclonal antibodies specific for lipopolysaccharide of Escherichia coli O26 and O111

Monoclonal antibody (MAb) 12F5 reacted with 35 Escherichia coli O26 isolates and cross-reacted with 1 of 365 non-E. coli O26 isolates. MAb 15C4 reacted with 30 E. coli O111 strains and 8 Salmonella O35 strains (possessing identical O antigen) but not with 362 other bacterial strains. Lipopolysaccharide immunoblots confirmed MAb O-antigen specificity.     

Modes of Escherichia coli Dps Interaction with DNA as Revealed by Atomic Force Microscopy

Multifunctional protein Dps plays an important role in iron assimilation and a crucial role in bacterial genome packaging. Its monomers form dodecameric spherical particles accumulating ~400 molecules of oxidized iron ions within the protein cavity and applying a flexible N-terminal ends of each subunit for interaction with DNA. Deposition of iron is a well-studied process by which cells remove toxic Fe²⁺ ions from the genetic material and store them in an easily accessible form. However, the mode of interaction with linear DNA remained mysterious and binary complexes with Dps have not been characterized so far. It is widely believed that Dps binds DNA without any sequence or structural preferences but several lines of evidence have demonstrated its ability to differentiate gene expression, which assumes certain specificity. Here we show that Dps has a different affinity for the two DNA fragments taken from the dps gene regulatory region. We found by atomic force microscopy that Dps predominantly occupies thermodynamically unstable ends of linear double-stranded DNA fragments and has high affinity to the central part of the branched DNA molecule self-assembled from three single-stranded oligonucleotides. It was proposed that Dps prefers binding to those regions in DNA that provide more contact pads for the triad of its DNA-binding bundle associated with one vertex of the protein globule. To our knowledge, this is the first study revealed the nucleoid protein with an affinity to branched DNA typical for genomic regions with direct and inverted repeats. As a ubiquitous feature of bacterial and eukaryotic genomes, such structural elements should be of particular care, but the protein system evolutionarily adapted for this function is not yet known, and we suggest Dps as a putative component of this system.     

Molecular Evolution of the Intimin Gene in O111 Clones of Pathogenic Escherichia coli

Intimin is an important virulence factor in two groups of enteric pathogens: enteropathogenic Escherichia coli (EPEC), which is a major cause of infant diarrhea in the developing world, and enterohemorrhagic E. coli (EHEC), which has caused large food-borne outbreaks of hemorrhagic colitis in the United States and other developed countries. Intimin is encoded on a 35-kb pathogenicity island called the locus of enterocyte effacement (LEE). At least five antigenic types have been described for the highly variable gene, and each type is generally characteristic of particular evolutionary lineages. We determined the nucleotide sequences of intimin and other LEE genes in two O111 clones that have not been amenable to typing. The sequences from both O111:H8 and O111:H9 differed from the Int-beta that is typical of other clones in the same evolutionary lineage. The sequence from the O111:H8 strains was a mosaic of divergent segments that alternately clustered with Int-alpha, Int-beta, or Int-gamma. The sequence from the O111:H9 clone consistently showed a close relationship with that from E2348/69, a distantly related strain that expresses Int-alpha. The results suggest that there have been multiple acquisitions of the LEE in the EHEC 2/EPEC 2 clonal lineage, with a recent turnover in either O111:H8 or its close relatives. Amino acid substitutions that alter residue charge occurred more frequently than would be expected under random substitution in the extracellular domains of intimin, suggesting that diversifying selection has promoted divergence in this region of the protein. An N-terminal domain that presumably functions in the periplasm may also be under positive selection.     

Production of a mannose-resistant fibrillar haemagglutinin by strains of Escherichia coli of EPEC serotype O111:H12

Two strains of Escherichia coli of serotype O111:H12 produced a mannose-resistant haemagglutinin (MREHA) of Duguid's pattern 7 that reacted strongly with the red cells of ox and sheep. These strains also adhered to HEp2-epithelial cells and formed fibrillae demonstrable by negative staining and immunogold labelling.     

Long-Term Survival of Shiga Toxin-Producing Escherichia coli O26, O111, and O157 in Bovine Feces

Cattle are an important reservoir of Shiga toxin-producing Escherichia coli (STEC) O26, O111, and O157. The fate of these pathogens in bovine feces at 5, 15, and 25°C was examined. The feces of a cow naturally infected with STEC O26:H11 and two STEC-free cows were studied. STEC O26, O111, and O157 were inoculated into bovine feces at 10¹, 10³, and 10⁵ CFU/g. All three pathogens survived at 5 and 25°C for 1 to 4 weeks and at 15°C for 1 to 8 weeks when inoculated at the low concentration. On samples inoculated with the middle and high concentrations, O26, O111, and O157 survived at 25°C for 3 to 12 weeks, at 15°C for 1 to 18 weeks, and at 5°C for 2 to 14 weeks, respectively. Therefore, these pathogens can survive in feces for a long time, especially at 15°C. The surprising long-term survival of STEC O26, O111, and O157 in bovine feces shows that such feces are a potential vehicle for transmitting not only O157 but also O26 and O111 to cattle, food, and the environment. Appropriate handling of bovine feces is emphasized.     

Prevalence and Virulence Factors of Escherichia coli Serogroups O26, O103, O111, and O145 Shed by Cattle in Scotland

A national survey was conducted to determine the prevalence of Escherichia coli O26, O103, O111, and O145 in feces of Scottish cattle. In total, 6,086 fecal pats from 338 farms were tested. The weighted mean percentages of farms on which shedding was detected were 23% for E. coli O26, 22% for E. coli O103, and 10% for E. coli O145. The weighted mean prevalences in fecal pats were 4.6% for E. coli O26, 2.7% for E. coli O103, and 0.7% for E. coli O145. No E. coli O111 was detected. Farms with cattle shedding E. coli serogroup O26, O103, or O145 were widely dispersed across Scotland and were identified most often in summer and autumn. However, on individual farms, fecal shedding of E. coli O26, O103, or O145 was frequently undetectable or the numbers of pats testing positive were small. For serogroup O26 or O103 there was clustering of positive pats within management groups, and the presence of an animal shedding one of these serogroups was a positive predictor for shedding by others, suggesting local transmission of infection. Carriage of vtx was rare in E. coli O103 and O145 isolates, but 49.0% of E. coli O26 isolates possessed vtx, invariably vtx₁ alone or vtx₁ and vtx₂ together. The carriage of eae and ehxA genes was highly associated in all three serogroups. Among E. coli serogroup O26 isolates, 28.9% carried vtx, eae, and ehxA—a profile consistent with E. coli O26 strains known to cause human disease.     

Cytoskeletal Asymmetrical Dumbbell Structure of a Gliding Mycoplasma,Mycoplasma gallisepticum,Revealed by Negative-Staining Electron Microscopy

Several mycoplasma species feature a membrane protrusion at a cell pole, and unknown mechanisms provide gliding motility in the direction of the pole defined by the protrusion. Mycoplasma gallisepticum, an avian pathogen, is known to form a membrane protrusion composed of bleb and infrableb and to glide. Here, we analyzed the gliding motility of M. gallisepticum cells in detail. They glided in the direction of the bleb at an average speed of 0.4 μm/s and remained attached around the bleb to a glass surface, suggesting that the gliding mechanism is similar to that of a related species, Mycoplasma pneumoniae. Next, to elucidate the cytoskeletal structure of M. gallisepticum, we stripped the envelopes by treatment with Triton X-100 under various conditions and observed the remaining structure by negative-staining transmission electron microscopy. A unique cytoskeletal structure, about 300 nm long and 100 nm wide, was found in the bleb and infrableb. The structure, resembling an asymmetrical dumbbell, is composed of five major parts from the distal end: a cap, a small oval, a rod, a large oval, and a bowl. Sonication likely divided the asymmetrical dumbbell into a core and other structures. The cytoskeletal structures of M. gallisepticum were compared with those of M. pneumoniae in detail, and the possible protein components of these structures were considered.Mycoplasmas are commensal and occasionally pathogenic bacteria that lack a peptidoglycan layer (50). Several species feature a membrane protrusion at a pole; for Mycoplasma mobile, this protrusion is called the head, and for Mycoplasma pneumoniae, it is called the attachment organelle (25, 34-37, 52, 54, 58). These species bind to solid surfaces, such as glass and animal cell surfaces, and exhibit gliding motility in the direction of the protrusion (34-37). This motility is believed to be essential for the mycoplasmas'' pathogenicity (4, 22, 27, 36). Recently, the proteins directly involved in the gliding mechanisms of mycoplasmas were identified and were found to have no similarities to those of known motility systems, including bacterial flagellum, pilus, and slime motility systems (25, 34-37).Mycoplasma gallisepticum is an avian pathogen that causes serious damage to the production of eggs for human consumption (50). The cells are pear-shaped and have a membrane protrusion, consisting of the so-called bleb and infrableb (29), and gliding motility (8, 14, 22). Their putative cytoskeletal structures may maintain this characteristic morphology because M. gallisepticum, like other mycoplasma species, does not have a cell wall (50). In sectioning electron microscopy (EM) studies of M. gallisepticum, an intracellular electron-dense structure in the bleb and infrableb was observed, suggesting the existence of a cytoskeletal structure (7, 24, 29, 37, 58). Recently, the existence of such a structure has been confirmed by scanning EM of the structure remaining after Triton X-100 extraction (13), although the details are still unclear.A human pathogen, M. pneumoniae, has a rod-shaped cytoskeletal structure in the attachment organelle (9, 15, 16, 31, 37, 57). M. gallisepticum is related to M. pneumoniae (63, 64), as represented by 90.3% identity between the 16S rRNA sequences, and it has some open reading frames (ORFs) homologous to the component proteins of the cytoskeletal structures of M. pneumoniae (6, 17, 48). Therefore, the cytoskeletal structures of M. gallisepticum are expected to be similar to those of M. pneumoniae, as scanning EM images also suggest (13).The fastest-gliding species, M. mobile, is more distantly related to M. gallisepticum; it has novel cytoskeletal structures that have been analyzed through negative-staining transmission EM after extraction by Triton X-100 with image averaging (45). This method of transmission EM following Triton X-100 extraction clearly showed a cytoskeletal “jellyfish” structure. In this structure, a solid oval “bell,” about 235 nm wide and 155 nm long, is filled with a 12-nm hexagonal lattice. Connected to this bell structure are dozens of flexible “tentacles” that are covered with particles 20 nm in diameter at intervals of about 30 nm. The particles appear to have 180° rotational symmetry and a dimple at the center. The involvement of this cytoskeletal structure in the gliding mechanism was suggested by its cellular localization and by analyses of mutants lacking proteins essential for gliding.In the present study, we applied this method to M. gallisepticum and analyzed its unique cytoskeletal structure, and we then compared it with that of M. pneumoniae.     

Optimization of enrichment and plating procedures for the recovery of Escherichia coli O111 and O26 from minced beef

AIM: Optimization of enrichment media and selective agars for the detection of Escherichia coli O26 and O111 from minced beef. METHODS AND RESULTS: This study compared a number of different enrichment conditions and plating media for the recovery of E. coli O26 and E. coli O111 from minced beef. The optimum enrichment conditions for E. coli O26 was observed in beef samples enriched at 41.5 degrees C in tryptone soya broth supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)). Similar enrichment conditions were optimal for E. coli O111 with the omission of potassium tellurite. The optimum agar for recovery of E. coli O26 and giving the most effective suppression of contaminants was MacConkey agar [lactose replaced by rhamnose (20 g l(-1))] and supplemented with cefixime (50 microg ml(-1)) and potassium tellurite (2.5 mg l(-1)). Optimum recovery of E. coli O111 was on chromocult agar, supplemented with cefixime (50 microg ml(-1)), cefsulodin (5 mg l(-1)) and vancomycin (8 mg l(-1)). Minced beef samples were inoculated with a number of strains of E. coli O26 (n=9) and O111 (n=8), and the developed enrichment and plating methods, used in combination with immunomagnetic separation, were shown to be an effective method for the recovery of all strains. CONCLUSIONS: Routine cultural methods for the recovery of E. coli O26 and O111 from minced beef are described. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimized enrichment and plating procedure described for the recovery of E. coli O111 and O26 from meat can be used to extend research on these emerging pathogens in beef.     

Discrimination of Escherichia coli O157, O26 and O111 from Other Serovars by MALDI-TOF MS Based on the S10-GERMS Method

Enterohemorrhagic Escherichia coli (EHEC), causes a potentially life-threatening infection in humans worldwide. Serovar O157:H7, and to a lesser extent serovars O26 and O111, are the most commonly reported EHEC serovars responsible for a large number of outbreaks. We have established a rapid discrimination method for E. coli serovars O157, O26 and O111 from other E. coli serovars, based on the pattern matching of mass spectrometry (MS) differences and the presence/absence of biomarker proteins detected in matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF MS). Three biomarkers, ribosomal proteins S15 and L25, and acid stress chaperone HdeB, with MS m/z peaks at 10138.6/10166.6, 10676.4/10694.4 and 9066.2, respectively, were identified as effective biomarkers for O157 discrimination. To distinguish serovars O26 and O111 from the others, DNA-binding protein H-NS, with an MS peak at m/z 15409.4/15425.4 was identified. Sequence analysis of the O157 biomarkers revealed that amino acid changes: Q80R in S15, M50I in L25 and one mutation within the start codon ATG to ATA in the encoded HdeB protein, contributed to the specific peak pattern in O157. We demonstrated semi-automated pattern matching using these biomarkers and successfully discriminated total 57 O157 strains, 20 O26 strains and 6 O111 strains with 100% reliability by conventional MALDI-TOF MS analysis, regardless of the sample conditions. Our simple strategy, based on the S10-spc-alpha operon gene-encoded ribosomal protein mass spectrum (S10-GERMS) method, therefore allows for the rapid and reliable detection of this pathogen and may prove to be an invaluable tool both clinically and in the food industry.     

Long-term survival of shiga toxin-producing Escherichia coli O26, O111, and O157 in bovine feces.

Cattle are an important reservoir of Shiga toxin-producing Escherichia coli (STEC) O26, O111, and O157. The fate of these pathogens in bovine feces at 5, 15, and 25 degrees C was examined. The feces of a cow naturally infected with STEC O26:H11 and two STEC-free cows were studied. STEC O26, O111, and O157 were inoculated into bovine feces at 10(1), 10(3), and 10(5) CFU/g. All three pathogens survived at 5 and 25 degrees C for 1 to 4 weeks and at 15 degrees C for 1 to 8 weeks when inoculated at the low concentration. On samples inoculated with the middle and high concentrations, O26, O111, and O157 survived at 25 degrees C for 3 to 12 weeks, at 15 degrees C for 1 to 18 weeks, and at 5 degrees C for 2 to 14 weeks, respectively. Therefore, these pathogens can survive in feces for a long time, especially at 15 degrees C. The surprising long-term survival of STEC O26, O111, and O157 in bovine feces shows that such feces are a potential vehicle for transmitting not only O157 but also O26 and O111 to cattle, food, and the environment. Appropriate handling of bovine feces is emphasized.     

Properties of a gonococcal inhibitor produced by Escherichia coli.

Strains of Escherichia coli can inhibit the in vitro growth of Neisseria gonorrhoeae. One E. coli strain released a potent agar-diffusible gonococcal growth inhibitor which was extracted and assayed in an agar well assay system. The culture conditions necessary to produce the inhibitor were determined. The inhibitor was bacteriostatic, in most cases, for N. gonorrhoeae. Based on ultrafiltration and column chromatography, the inhibitor appeared to have a molecular weight in the range of 1200 to 2000. Evidence that the molecule contained charged sites was obtained by membrane binding and column chromatography. The inhibitor was stable to extremes of heat, cold and pH. It was not volatile or susceptible to proteolytic enzymes, lysozyme, lipase, DNAase, RNAase or certain chelating agents. Its activity was completely blocked by ferric ammonium citrate. This inhibitor is dissimilar to previously reported gonococcal inhibitors of bacterial origin.