Virulence plasmid-associated sensitivity to rifampicin and novobiocin in Escherichia coli

Introduction of the ColV, I-K94 or ColV-K30 plasmid into Escherichia coli K12 resulted in increased sensitivity to growth inhibition and killing by rifampicin and novobiocin. For increased rifampicin sensitivity, both transfer and colicin components had to be present whereas transfer components plus another unidentified ColV component were needed for novobiocin sensitivity. The Co1 factor Co1B-K98 conferred increased rifampicin sensitivity, an effect not dependent on transfer components, but this plasmid had no effect on the novobiocin sensitivity of E. coli. The effects of ColV, I-K94 on rifampicin sensitivity were fully reversed by magnesium ions but Mg²⁺ had only a slight effect on the novobiocin inhibition of the Co1V, I-K94⁺ strain and the rifampicin inhibition of the Co1B-K98⁺ strain. Prior growth at 25C greatly reduced the effects of Co1V, I-K94 on rifampicin and novobiocin sensitivity but had essentially no effect on rifampicin sensitivity of the Co1B-K98⁺ strain.     

Contributions of traT and iss genes to the serum resistance phenotype of plasmid ColV2-K94

Abstract A genetic determinant for serum resistance, designated iss , has been found previously on the colicinogenic plasmid ColV2-K94. In this work we have identified a second serum resistance gene, traT , on ColV2-K94. The serum resistance mediated by derivatives of ColV2-K94 was due to presence of one or both of the iss and traT genes. Plasmid pWS12 (TraT⁺ Iss⁺) contained the kanamycin (Km) resistance transposon Tn 903 inserted near the origin of replication of ColV2-K94, and plasmids pWS15 (TraT⁺), pWS16 (TraT⁺) and pWS18 (TraT⁺ Iss⁺) were deletion derivatives of pWS12 constructed in vitro and in vivo. pWS12 and pWS18 conferred a 20-fold increase in relative resistance to 20% guinea pig serum when introduced into the serum-susceptible, genetically defined recA strain of Escherichia coli K-12, AB2463. Plasmids pWS15 and pWS16, from which iss had been deleted, still conferred 5-fold increases in relative resistance on AB2463. The level of resistance conferred on this strain by the antibiotic resistance plasmid R100–1 (which expresses the traT serum resistance gene) was comparable to that of plasmids pWS15 and pWS16. The 25-kDa traT gene product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the outer membrane proteins of strain AB2463 carrying ColV2-K94. This protein cross-reacted immunologically with the traT protein expressed by F or R100–1. Our results indicated that both traT and iss are capable of mediating serum resistance in ColV2-K94.     

Mobilization of CS fimbriae-associated plasmids of enterotoxigenic Escherichia coli of serotype O6: K15: H16 or H- into various wild-type hosts

Abstract CS fimbriae-associated plasmids of two enterotoxigenic Escherichia coli strains of serotype O6: K15: H16 or H- (biotypes A and F) with M _r values of 51 × 10⁶ and 72 × 10⁶, respectively, were mobilized into various alternative host bacteria. Expression of CS1 or CS2 fimbriae was obtained when either of the CS fimbriae-associated plasmids was introduced into CS Fim⁻, O6: K15: H16 or H- recipients with rhamnose-negative and rhamnose-positive fermentation phenotypes, respectively, whereas CS3 fimbriae were expressed irrespective of the biotype of the recipient. On transfer into a CS Fim⁻ variant of an enterotoxigenic O8: H9 strain and into two K-12 strains, a CS3-fimbriae-only phenotype was conferred by the presence of either of the plasmids. When a CS Fim⁻ variant of a Rha⁺ CS2-fimbriae-only strain of serotype O6: K15: H16 harboured either of the plasmids, both CS2 and CS3 fimbriae were expressed, indicating that the rare CS2-fimbriae-only wild-type phenotype is probably due to the presence of a defective plasmid in such strains. Mobilization of the 51 MDa CS fimbriae-associated plasmid into five non-enterotoxigenic Rha⁺ porcine isolates of E. coli with O6 serotypes other than O6: K15: H16 or H- yielded CS3-fimbriae-only transconjugants. Thus the correlation between a Rha⁺ fermentation phenotype and expression of CS2 fimbriae does not hold in general for O-group 6 strains.     

Fibronectin binding and cell-surface hydrophobicity of attaching effacing enteropathogenic Escherichia coli strains isolated from newborn and weanling rabbits with diarrhoea

Abstract 32 different strains of Escherichia coli isolated from rabbits with diarrhoea were studied for cell-surface properties which may be involved in intestinal colonisation. Strains isolated from diarrhoeic suckling (6 strains) and weaning (26 strains) rabbits which were shown to attach to brush borders in vivo, showed high relative cell-surface hydrophobicity as determined by the Salt Aggregation Test (SAT) when grown on Colonisation Factor Antigen (CFA) agar at 33°C. Cells of these strains grown to express surface hydrophobicity were also defined as high, moderate or low binders of ¹²⁵I-fibronectin or its ¹²⁵I-29-kDa fragment in a standard binding assay. Based on these findings, we propose that binding to intestinal cell surface (mucus)-associated fibronectin may be an early important step in intestinal colonisation of the small bowel in enteropathogenic E. coli (EPEC) diarrhoea in rabbits and other animal species.     

Habituation to normally lethal acidity by prior growth of Escherichia coli at a sub-lethal acid pH value

Both Col^- and ColV, I-K94⁺ strains of Escherichia coli , grown at pH 7–0, failed to grow after relatively short periods of exposure to pH 3·0 or 3·5. After growth in exposure medium initially at pH 5·0, both strains were almost unaffected by exposure to such acid pH values. Addition of catalase to nutrient agar only slightly increased plating efficiency after acid treatment and very slightly reduced the difference in survival, after acid treatment, between organisms grown from pH 5·0 and those grown from pH 7·0. Accordingly, acid resistance of organisms grown from pH 5·0 is not chiefly due to greater resistance to hydrogen peroxide already present in nutrient media.     

Virulence plasmid-associated adhesion of Escherichia coli and its significance for chlorine resistance

Introduction of the ColV, I-K94 virulence plasmid into strains of Escherichia coli led (for four out of five strains tested) to a marked increase in the ability of organisms to adhere to glass beads. For strain 1829, the plasmid led to increased attachment to other materials including sand, agar, agarose, chitin and cellulose. The increased adhesion to glass beads was due to the presence of the plasmid and not to its introduction into a variant with altered adhesive properties. The plasmid-encoded VmpA protein did not appear to be necessary for the ColV, I-K94-promoted adhesion but adhesion was absolutely dependent on the presence of derepressed levels of transfer components in the ColV+ strains and partially dependent on the presence of colicin components. The extent of the plasmid-promoted adhesion was greatest for organisms grown at 30 degrees, 37 degrees or 42 decrees C and adhesion was almost abolished by growth at 21 degrees or 25 degrees C; this finding is in accord with transfer and colicin components being involved in adhesion. Of several other plasmids tested for their effects on adhesion, those with derepressed transfer properties showed a marked effect as did the RI resistance plasmid. Because of the ease of handling glass bead-attached organisms, such preparations were used as a model for studying the relevance of attachment to the resistance of E. coli to chlorination in the water purification process. Organisms of 1829 ColV, I-K94, attached to glass beads, were more resistant to damage and killing by chlorine than were unattached organisms. Three findings suggest that such chlorine resistance may be significant for survival during water chlorination. Firstly, ColV, I-K94+ bacteria became attached if incubated in sewage effluent with glass beads at 20 degrees C. Secondly, ColV+ organisms already attached to glass beads maintained their attachment during 24 h incubation in effluent at 20 degrees C and thirdly such effluent incubated organisms remained chlorine resistant provided that they retained their attachment.     

Copper sensitivity in an envelope mutant of Escherichia coli and its suppression by ColV, I-K94

A phage K3-resistant isolate from Escherichia coli P678-54 was devoid of both the OmpA and OmpC proteins but had high levels of the OmpF protein. Associated with these changes, the strain showed increased sensitivity to inhibition by detergents and greatly increased sensitivity to Cu²⁺. Introduction of the ColV, I-K94 plasmid into this mutant produced a derivative with markedly increased resistance to Cu²⁺ ions but unchanged detergent sensitivity. Analysis of membranes showed that the ColV, I-K94⁺ derivative had essentially no OmpF protein in its outer membrane. A ca 36 K outer membrane protein was present which resembled the OmpC protein in size and failure to dissociate in SDS at low temperature. It was distinct from the OmpC protein, however, in failing to allow either tetracycline uptake or the adsorption of T4-type phages. The possible significance of OmpF porin derepression (and its reversal by ColV, I-K94) for enterobacterial survival in aquatic situations is discussed.     

Fate of pathogenic and non-pathogenic Escherichia coli strains in two fermented milk products

F eresu , S. & N yati , H. 1990. Fate of pathogenic and non-pathogenic Escherichia coli strains in two fermented milk products. Journal of Applied Bacteriology 69 , 814–821.
The growth and survival of pathogenic and non-pathogenic strains of Escherichia coli was determined in traditionally fermented pasteurized and unpasteurized milk and in Lacto, an industrially fermented milk. Each milk treatment was incubated at 20C for 24 h and then stored at either 20C or 5C for 96 h.
Lacto inhibited all the three E. coli strains. Two strains could not be recovered and the third survived only in very low numbers after 24 h storage of Lacto at both 20C and 5C. All three E. coli strains survived and multiplied to maximum cell numbers in the range 10⁷-10⁹/ml during traditional fermentation of unpasteurized milk. Cell numbers decreased to 10³-10⁶ and 10²-10⁵ during storage of the fermented product at 20C and 5C respectively. Higher maximum numbers, 10⁹-10¹⁰, of the three strains of E. coli were attained during traditional fermentation of pasteurized milk. The numbers decreased to 10⁵-10⁸ and 10⁴-10⁷ during storage of the fermented product at 20C and 5C respectively. Generally, fewer E. coli survived when the fermented milk products were stored at refrigeration temperature.     

Chilling-induced potassium leakage of cultured citrus cells

Callus of 'Marsh' grapefruit ( Citrus paradisi Macf.) albedo tissue was used to investigate the effect of preconditioning temperature on the rate of chilling - stimulated K⁺ leakage. Callus grew most rapidly at 30°C and attained a weight of about 1 g after 30 days. The rate of K.⁺ leakage from nonchilled callus tissue decreased as temperature decreased from 20 to 7.5°C, but no measurable change in rate was observed between 7.5 and 0°C. When calli were held for 40 days at 0¹ 2.5 or 5°C, K⁺ leakage increased 200%, 60% or 0%) respectively. Holding callus for 5 days at 10 or 15°C prior to chilling for 40 days at 0°C prevented the increase in K⁺ leakage observed in callus receiving no preconditioning treatment. Preconditioning at 7.5 and 20°C was less effective in reducing chilling - induced leakage. Preconditioning at 10°C for 5, 2 or 1 day reduced chilling – induced leakage after 40 days at 0°C by 50%, 33% and 15%. respectively.     

Behaviour of Staphylococcus aureus strains, producers of enterotoxins C1 or C2, during the manufacture and storage of Burgos cheese

Burgos cheese was manufactured from pasteurized ewes milk inoculated with Staphylococcus aureus strains FRI 137 and FRI 361, at levels of ca 10³ and 10⁵ cfu/ml and stored at 4°, 10° and 15°C and at room temperature (10°-15°C). Populations of Staph. aureus and mesophilic aerobes, pH, and production of thermonuclease and enterotoxins C₁ and C₂ were investigated. Aerobic counts increased during cheese-making and storage. With both test strains, important growth was observed only during the storage period, the larger levels corresponding to the higher temperatures. Although Staph. aureus strains attained populations of over 10⁸ cfu/g, no enterotoxin was detected. Strain FRI 361 reached 10⁷ cfu/g without production of a detectable amount of thermonuclease. With strain FRI 137, the minimal population associated with enzyme activity was influenced by the inoculum size. Staphylococcus aureus counts are better indicators of staphylococcal growth in Burgos cheese than the thermonuclease test.     

The effect of moisture potential on growth and survival of root nodule bacteria in peat culture and on seed

Peat from three sources was dried, milled and packed separately in polyethylene bags and sterilized by irradiation. The carrier was impregnated with broth cultures of either Rhizobium leguminosarum bv. trifolii strain WU95, Bradyrhizobium japonicum strain CB1809 or B. lupini strain WU425 and sterile water to provide five moisture potentials in the range > - 1 × 10⁴ - 1 × 10⁶ Pa. The packets were stored at 26°C under conditions which restricted moisture loss. Numbers of root nodule bacteria were counted at intervals up to 12 weeks. No single moisture potential was optimum for all strains in all carriers because of a significant ( P < 0.05) interaction between moisture potential × strain × carrier × time. Where direct comparisons could be made, all strains survived best at - 1 × 10⁴ and/or −3.2 × 10⁴ Pa. Seeds of Trifolium subterraneum and polypropylene beads (used to avoid seed coat toxicity), were inoculated with WU95 prepared in two sources of peat and at each of the above moisture potentials and stored at 15°C. Seed coat toxicity significantly effected the log death rate ( k ) of WU95 on subterraneum clover seed for the period 0–0.25 d ( k 1.796) compared with k - 0.399 for polypropylene beads. In the first 24 h moisture did not affect survival but by 28 d rhizobia grown in Badenoch peat survived best at −3.2 × 10⁴ Pa. In Millicent peat, survival was equally as good at −3.2 × 10⁴ and −1 × 10⁴ Pa.     

Recombinant DNA transfer to Escherichia coli of human faecal origin in vitro and in digestive tract of gnotobiotic mice

Abstract The role of helper elements in the mobilisation of pBR recombinant plasmids ( tra ⁻, mob ⁻, ori T⁺ and tra ⁻, mob ⁻, ori T⁻) from genetically engineered Escherichia coli K12 strains to other K12-strains and to wild-type E. coli strains of human faecal origin was examined. Transfer experiments were done in the digestive tract of axenic (germ free) and gnotobiotic mice, associated with human faecal flora, HFF. The kinetics of implantation of donors, recipients and transconjugants were determined. Mobilisation of ori T⁺ pBR-type plasmids, by trans-complementation with the products of tra and mob genes was obtained with E. coli K12, in the digestive tract of axenic mice and the resulting transconjugants became established together with the recipient and donor strains. Such mobilisation was only observed sporadically with one E. coli of human origin in axenic mice, but did not occur in gnotobiotic HFF mice. The E. coli strains of human origin were able to promote transfer of an ori T⁻ pBR-type plasmid in vitro but not in axenic or gnotobiotic mice. Transconjugants of wild-type strains obtained in in vitro mating experiments and inoculated into gnotobiotic HFF mice were eliminated as rapidly as the recombinant K12 strains. This work indicates that ≥ 50% of wild-type E. coli strains were able to promote transfer of pBR ori T⁻ plasmids in vitro.     

Cations Affect [3H]Mazindol and [3H]WIN 35,428 Binding to the Human Dopamine Transporter in a Similar Fashion

Abstract: The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn²⁺, Mg²⁺, Hg²⁺, Li⁺, K⁺, and Na⁺ were assessed on the binding of [³H]mazindol and [³H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn²⁺ (30–100 µ M ) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [³H]mazindol; Mg²⁺ (0.1–100 µ M ) had no effect; Hg²⁺ at ∼3 µ M stimulated binding to membranes, with a relatively smaller effect for [³H]mazindol than [³H]WIN 35,428 at 0°C, and at 30–100 µ M inhibited both intact cell and membrane binding; Li⁺ and K⁺ substitution (30–100 m M ) inhibited binding to membranes more severely than to intact cells; and Na⁺ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn²⁺ at 0 and 21°C and Hg²⁺ at 0°C.     

Growth and accumulation of N, K+, Ca2+ and Mg2+ in barley exposed to various nutrient regimes and root/shoot temperatures

Six cultivars of spring barley ( Hordeum vulgare L. cvs Salve, Nümberg II, Bomi, Risø 1508, Mona and Sv 73 608) were grown in water culture for three weeks with various combinations of mineral supply and differential roots/shoot temperatures during the growth period. Most important for growth and accumulation of N, K⁺, Ca²⁺ and Mg²⁺ was the mineral supply, followed by the root temperature and the choice of cultivar. Treatments with low mineral supply or low root temperature induced a uniform reduction in growth and accumulation of the ions studied. The effects of low mineral supply and low root temperature on growth and N accumulation was additive, which indicates that these factors exert their influence independently of each other.
Roots grown at 10°C were smaller and Rb⁺(⁸⁶Rb) influx was higher than in roots grown at 20°C. It is suggested that the control of Rb⁺(⁸⁶Rb) influx is affected by the root temperature and the age of the plants. The higher ⁸⁶Rb⁺ (⁸⁶Rb) influx into the low temperature roots could not compensate for the smaller root size. However, the lower total mineral accumulation made up for the needs of the smaller plants and cannot explain the reduction in growth.     

Col V plasmids and the resistance of Escherichia coli to divalent copper ions

Free organisms of both plasmid-free and plasmid-carrying strains of Escherichia coli were killed by incubation in water containing low levels of cupric ions. Sensitivity was temperature-dependent with killing being more marked at 20° or 25°C than at 10° or 15°C. In contrast to the effects of other inhibitors from natural waters (which affect free Col V⁺organisms more than Col^-ones), free Col^-and Col V⁺organisms were equally sensitive to kill by Cu²⁺. Attachment to glass beads essentially abolished sensitivity to cupric ions with full survival after exposure to 15 μ g/ml. This applied to both p⁺and p^-strains but attachment would have more effect on the survival of p⁺organisms in natural waters because some plasmids markedly enhance attachment.     

Effect of growth temperature on virulence of strains of Listeria monocytogenes in the mouse: evidence for a dose dependence

Growth of Listeria monocytogenes at 4°C significantly increased its virulence for mice by the intravenous route and the effect was dose-dependent. Virulence was apparent only at a dose of about or above 10⁴ viable listerias. At slightly lower doses of about 10³, no such effect was observed. Growth at 4°C did not increase the virulence of the strains for mice by oral-gastric challenge when given at doses of approximately 10¹⁰.     

Instability of plasmid-encoded citrate permease in Leuconostoc

M. KIHAL, H. PRÉVOST, M.E. LHOTTE, D.Q. HUANG AND C. DIVIÈS. 1996. The conversion from citrate positive (Cit⁺) to citrate negative (Cit^-) phenotype of six strains of Leuconostoc mesetiteroides was followed during growth in milk and buffered or unbuffered MRS medium at 30 or 37°C. High rate of loss of Cit⁺ phenotype was observed. The Cit^- phenotype was found to be linked to the loss of 22 to 23 kb plasmids. All Cit^- mutants isolated from Leuc. mesenteroides subsp. cremoris 195 reverted spontaneously to the Cit⁺ phenotype. Hybridization experiments using a 0.8 kb fragment of the citP gene of Leuc. mesenteroides showed that all the plasmids which were lost in Cit^- mutants encoded for a citrate permease. However, neither plasmid nor genomic DNA from Leuc. mesenteroides subsp. cremoris 195 hybridized with the citP probe.     

Oxygen consumption in Caridina nilotica (Decapoda: Atyidae) in relation to temperature and size

SUMMARY. The oxygen consumption of shrimps ranging from 1 to 30 mg dry mass was determined at 18, 24 and 30°C using a continuous flow recording respirometer based upon a Clark-type oxygen electrode. Respiration (ascribed to routine metabolism) is described by the power curve: R = a M^b , ( R =μg O₂ h⁻¹, M = mg dry mass), which gives values of a = 1.632, 2.564 and 4.181, and b = 0.800, 0.898, and 0.793, at 18, 24 and 30°C respectively. The single expression, R = 0.008 T ^1.829 M ^0.830 provides a reasonable prediction of respiration as a combined function of shrimp size ( M ) and temperature (T, °C). Using an energy equivalent of 14.14 J mg O₂⁻¹ estimates of the energy requirements ( E , J h⁻¹ 10⁻³) of routine metabolism are given by the expression: E = 0.115 T ^1.829 M ^0.830.
Variability in oxygen consumption values between individuals is discussed and the observations on C. nilotica are compared with other crustacean studies.     

Combining heat treatment and subsequent incubation temperature to prevent growth from spores of non-proteolytic Clostridium botulinum

Refrigerated processed foods of extended durability rely on a mild heat treatment combined with refrigerated storage to ensure microbiological safety and quality. The principal microbiological safety risk in foods of this type is non-proteolytic Clostridium botulinum. In this article the combined effect of mild heat treatment and refrigerated storage on the time to growth and probability of growth from spores of non-proteolytic Cl. botulinum is described. Spores of non-proteolytic Cl. botulinum (two strains each of type B, E and F) were heated at 90°C for between 0 and 60 min and subsequently incubated at 5°, 10° or 30°C in PYGS broth in the presence or absence of lysozyme. The number of spores that resulted in turbidity depended on the combination of heat treatment, incubation time and incubation temperature they received. Heating at 90°C for 1 or more min ensured a 10⁶ reduction when spores were subsequently incubated at 5°C for up to 23 weeks. Heating at 90°C for 60 min ensured a 10⁶ reduction over 23 weeks when subsequent incubation was at 10°C in the presence of added lysozyme. The same treatment did not reduce the spore population by 10⁶ when subsequent incubation was at 30°C.     

Isolation and characterization of large lactococcal phage resistance plasmids by pulsed-field gel electrophoresis

Abstract Five phage-resistant Lactococcus lactis strains were able to transfer by conjugation the lactose-fermenting ability (Lac⁺) to a plasmid-free Lac⁻ L. lactis strain. In each case, some Lac⁺ transconjugants were phage-resistant and contained one or two additional plasmids of high molecular mass, as demonstrated by pulsed-field gel electrophoresis. Plasmids pPF144 (144 kb), pPF107 (107 kb), pPF118 (118 kb), pPF72 (72 kb) and pPF66 (66 kb) were characterized: they are conjugative (Tra⁺), they confer a phage-resistant phenotype and they bear lactose-fermenting ability (Lactose plasmid) except for the last two. Plasmids pPF144, pPF107 and pPF118 resulted probably from a cointegrate formation between the Lactose plasmid and another plasmid of the donor strain, whereas pPF72, pPF66 and the Lactose plasmid were distinct in the corresponding transconjugants. Plasmids pPF72 and pPF66 produced a bacteriocin. At 30°C, the phage resistance conferred by the plasmids was complete against small isometric-headed phage and partial against prolate-headed phage, except for pPF107 whose phage resistance mechanism was totally effective against both types of phages, but was completely inactivated at 40°C. Restriction maps of four of the plasmids were constructed using pulsed-field gel electrophoresis.