Bacteriophage resistance of attached organisms as a factor in the survival of plasmid-bearing strains of Escherichia coli

Unattached organisms of plasmid-free and plasmid-bearing strains of Escherichia coli showed marked sensitivity to phages T4, Tula and K3 on incubation in broth at 37°C but organisms attached to glass beads or sand were resistant. Phage T4 sensitivity of free organisms and resistance of glass bead-attached ones were also observed when incubation was in broth at 15° or 20°C or anaerobically at 37°C. In contrast, free and attached organisms were resistant at 37°C or lower temperature when incubation was in poor media. It seems likely that the presence of phage will be a major factor reducing survival in the intestine and in sewage and that attachment (which is more significant for strains bearing certain plasmids) will protect. In contrast, survival of either free or attached organisms in polluted water will probably not be significantly influenced by the presence of phage.     

Effect of growth temperature on complement-mediated killing of mesophilic Aeromonas spp. serotype O:34

Abstract Mesophilic Aeromonas spp. strains (serotype O:34) showed sensitivity to complement-mediated killing when they were cultivated at 37°C (serum-sensitive) but not when they were cultivated at 20°C (serum-resistant). These strains produced smooth lipopolysaccharide when they were grown at 20°C and rough lipolysaccharide when cultivated at 37°C. The reason for the resistance to complement-mediated killing could be that C3b is rapidly degraded (possibly because it is bound far from the cell membrane), consequently the lytic complex (C5b-9) is not formed.     

Studies on the Multiplication of Salmonellae in Various Enrichment Media at Different Incubation Temperatures

Several selected Salmonella strains did not multiply in tetrathionate brilliant green bile medium when the inoculum was small and the medium was incubated at 43° C. Gradual heating from 20° to 43° C and dilution of the medium with buffered peptone water (1:10) containing egg yolk did not decrease its inhibitory properties. It became less inhibitory, however, after the growth of other Enterobacteriaceae. These findings stress the advantage of using non-selective pre-enrichment since in that case the Muller-Kauffmann tetrathionate broth will be inoculated with a large number of salmonellae and other Enterobacteriaceae thus facilitating the isolation of the former.     

Reversible change in embryonic diapause intensity by mild temperature in the Chinese rice grasshopper, Oxya chinensis

The effects of temperature on maintenance and termination of embryonic diapause were investigated in Jining (35.4°N, 116.6°E) and Sihong (33.5°N, 118.2°E) strains of the Chinese rice grasshopper, Oxya chinensis Thunberg (Orthoptera: Catantopidae). Eggs of both strains entered diapause when incubated at 30, 25, or 20 °C. Chilling at 8 °C had an evident effect on diapause termination and almost all eggs chilled for 60 days ended diapause development. Chilling of eggs at 8 °C for only 20 days failed to result in any hatching at 20 °C, suggesting that such level of chilling was not enough to induce diapause termination. However, the treatment combining incubation of eggs at 30 °C for varying lengths of time with subsequent incubation to 20 °C had a distinct effect on the completion of diapause of the eggs. The results indicate that there were two temperature optima, that is, low temperature (chilling) and high temperature, for diapause development in this grasshopper species. Incubation of chilled eggs at 20 °C for 5–15 days followed by further incubation at 25 °C reduced termination of diapause significantly compared with the eggs only chilled at 8 °C. Exposure of eggs chilled at 8 °C to a pulse of 25 °C from 1 to 7 days, separated by a 20-day interval at 8 °C, resulted in a decrease in the percentage of successfully hatched eggs as the length of the pulse of 25 °C increased. The results suggest that diapause intensity may be restored at moderately high temperatures. This reversible change in diapause intensity would play an important role in maintaining diapause before winter.     

The effects of phosphate supply on uptake of potassium ions, 2,4-D and atrazine by wheat and maize

The germination percentage of peach [ Prunus persica (L.) Batsch cv. Halford] seeds at 20°C was low (< 20%) after incubation at 5°C for as long as 35 days, but then increased considerably (> 40%) when the seeds were maintained at 5°C for longer than 42 days. Four zones of gibberellin-like activity were found in partially purified seed extracts. Gibberellin-like activity remained low in seeds incubated at 5°C for as long as 28 days, but increased significantly in three of these zones after 35 days, and in the fourth zone after 49 days. The increase in gibberellin-like activity was evident prior to the transfer of the seeds to 20°C. Moreover, seeds maintained at 5°C germinated at this temperature after 63 days. For seeds incubated and germinated at 20°C, both the germination percentage and the gibberellin-like activity remained low throughout the experimental period. Application of the growth retardant paclobutrazol to seeds after 28 days of a 49 day total incubation period at 5°C did not substantially reduce seed germination, although the increase in gibberellin-like activity was prevented. Seeds did, however, require a longer time to germinate after transfer to 20°C and were dwarfed in appearance. Application of GA₃ to seeds prior to stratification increased the percentage germination of seeds only when they had been incubated at 5°C for at least 35 days. The major changes in gibberellin-like activity are, therefore, associated not so much with the processes which allow germination to take place in peach, but more with those processes which allow normal growth and development of the seedling.     

Nitrogenase activity (acetylene reduction) in root-associated, cold-climate species of Azospirillum, Enterobacter, Klebsiella and Pseudomonas growing at various temperatures

Abstract 23 Strains of diazotrophic root-associated bacteria isolated from various parts of Finland were tested for nitrogenase activity during growth at various temperatures. Nitrogenase activity was optimal at 20–37°C in cultures of Klebsiella pneumoniae , and at 14–20°C in cultures of Klebsiella terrigena and Enterobacter agglomerans . Strains of K. terrigena and E. agglomerans showed no activity at 37°C, and K. pneumoniae only minimal or no activity at 14°C. Azospirillum lipoferum exhibited high nitrogenase activity at both 28–37°C, but less than 25% of optimal activity at 20°C and no activity at 14°C. Pseudomonas sp. expressed nitrogenase activity at 14–28°C. None of the strains manifested nitrogenase activity at 4 or 42°C. There were only small local variations within a species between strains isolated at different locations.     

Incidence and Spoilage Potential of Isolates from Vacuum-packaged Meat of High pH Value

Meat of high pH value (6·6) showing dark-cutting characteristics was vacuum-packaged and stored for up to 8 weeks at 0–2°C. 'Off'-odours were detected on opening the packages after 6 weeks of storage. Total counts at this stage were ca. 10⁷/cm² of which lactobacilli were the major component, with ca. 10⁶/cm² Gram negative organisms. Psychrotrophic Enterobacteriaceae represented a major proportion of the microflora only after the full 8 weeks of storage and were not detected previously. Aerobic storage of steaks cut from the vacuum packaged meat stored for 8 weeks resulted in a predominantly Gram negative spoilage flora.
Inoculation studies on meat of normal pH value (5·4) and appearance using representative isolates from the vacuum-packaged meat microflora indicated that most of the test organisms were capable of causing spoilage under aerobic conditions but few under vacuum-packaging when incubated at 4°C. On meat of higher pH value (6·15) many of the Gram negative isolates did not grow as well, whereas the Gram positive isolates grew better than on meat of normal pH value when held under aerobic conditions. Under vacuum-packaging all but one isolate grew as well or better on meat of high pH value than on normal meat at 4°C and objectionable odours were more marked.     

Toxicity of crude extracellular products of Aeromonas hydrophila in tilapia, Tilapianilotica

Extracellular products (ECP) secreted from Aeromonas hydrophila with haemolytic andproteolytic activity were studied with respect to temperature and time of incubation as well as thelethal toxicity on tilapia, Tilapia nilotica . The highest production of the haemolysin productwas achieved when Aer. hydrophila was grown at 35°C for 30 h. Tilapia erythrocytewas found to be more susceptible than sheep erythrocyte for determining the haemolytic activity.The haemolytic activity against tilapia erythrocyte was completely inactivated after heating theECP at 60°C for 10 min or 55°C for 15 min. The proteolytic activity was maximized whenthe bacterium was grown at 30°C for 36 h. Complete inactivation of the protease enzyme wasperformed after heating the ECP at 80°C for 10 min or 70°C for 15 min. Aeromonashydrophila was found to produce haemolytic and proteolytic exotoxin lethal to tilapia (LD₅₀ 2·1 × 10⁴ cell/fish), as well as heat stable unknown virulent factors thatwere responsible for 20% mortality. The lethality of ECP was decreased by heating andcompletely inactivated by boiling at 100°C for 10 min.     

Antibacterial activity of the lactoperoxidase system against Aeromonas hydrophila in broth, skim milk and ewes' milk

The effect of the lactoperoxidase (LP)-system on Aeromonas hydrophila in broth, skim milk and ewes' milk was investigated. The behaviour of three strains in activated and control substrates was studied at 6 and 28°C. The activated ovine lactoperoxidase was bactericidal for Aer. hydrophila . In the three LP-treated substrates the tested strains were completely inactivated after 2 (28°C) and 24 h (6°C) and then remained undetectable during storage for up to 25 d. The use of the LP-system as a preservative against Aer. hydrophila could be very useful even when adequate cooling facilities are available.     

Psychrotrophic bacterial flora of raw ewes'milk, with particular reference to Gram negative rods

The microbial flora of 141 samples of raw ewes'milk was determined, before and after storage for 72 h at 4° and 7°C. Penicillin-resistant bacteria represented ca 61% of 1760 psychrotrophic isolates from refrigerated milk samples. Pseudomonas fluorescens and Pseudomonas fluorescent group-related strains predominated ( ca 86%) in the Gram negative psychrotrophic microflora. Leuconostoc dextranicum was the most frequent Gram positive psychrotrophic species isolated.     

SEASONALITY AND THERMAL ACCLIMATION OF REACTIVE OXYGEN METABOLISM IN FUCUS VESICULOSUS (PHAEOPHYCEAE)

The intertidal brown macroalga Fucus vesiculosus L. acclimates its defense against reactive oxygen in response to both (1) growth at different temperatures in laboratory culture and (2) seasonal changes in environmental conditions. Fucus vesiculosus was grown in seawater at 0° C, 20° C, and at 0° C with a 3-h daily emersion at −10° C. Algae grown at low temperature, both with and without freezing, produced less reactive oxygen after severe freezing stress than those grown at 20° C. These differences were correlated with growth temperature-induced changes in activities of superoxide dismutase (SOD), glutathione reductase, and ascorbate peroxidase. The contents of tocopherols increased with increased cultivation temperature, whereas the activity of catalase and the content of glutathione and ascorbate did not change. Growth at 0° C increased the resistance of photosynthesis to freezing and reduced photoinhibition in high light at 5° C; the latter effect was further increased in algae subject to daily freezing. These data suggest that elevated activity of reactive oxygen scavenging enzymes, especially SOD, increases the resistance to photoinhibition, at least at low temperature, as well as being important for freezing tolerance. Seasonal changes in reactive oxygen metabolism showed a similar pattern to those elicited by temperature in laboratory culture. Summer samples had lower activities of most reactive oxygen scavenging enzymes than algae collected in autumn and winter when water temperatures were lower. In contrast to the laboratory experiments, ascorbate content did change and was lower during the winter than summer, whereas the content of glutathione was not influenced by season. Overall, the data not only indicate that temperature plays an important role in the regulation of stress tolerance and reactive oxygen metabolism but also suggest that other factors are also involved.     

Low temperature delays development of photosystem II activity in winter rye leaves

The ability of leaves to acclimate photosynthetically to low temperature was examined during leaf development in winter rye plants ( Secale cereale L. cv. Puma) grown at 20°C or at 6°C. All leaves grown at 6°C exhibit increased chlorophyll (Chl) levels per leaf area, higher rates of uncoupled, light-saturated photosystem I (PSI) electron transport, and slower increases in photosystem II (PSII) electron transport capacity, when compared with 20°C leaves. The stoiehiometry of PSI and PSII was estimated for each leaf age class by quantifying Chl in elcctrophorctic separations of Chl-protein complexes. The ratio of PSII/PSI electron transport in 20°C leaves is highly correlated with the ratio of core Chl a -proteins associated with PSII (CPa) to those associated with PSI (CP1). In contrast, PSII/PSI electron transport in 6°C leaves is not as well correlated with CPa/CP1 and is related, in part, to the amount and organization of light-harvesting Chl a/b -proteins associated with PSII. CPa/CP1 increases slowly in 6°C leaves, although the ratio of CPa/CP1 in mature 20°C and 6°C leaves is not different. The results suggest that increased PSI activity at low temperature is not related to an increase in the relative proportion of PSI and may reflect, instead, a regulatory change. Photosynthetic acclimation to low environmental temperature involves increased PSI activity in mature leaves shifted to 6°C. In leaves grown entirely at 6°C, however, acclimation includes both increased PSI activity and modifications in the rate of accumlation of PSII and in the organization of LHCII.     

Mycotoxin production by some wood-associated Penicillium spp.

Twenty-five strains of Penicillium spp. isolated from mould-infected discoloured outdoor softwood were analysed for mycotoxin production. Patulin was found to be produced by Penicillium expansion at 4° and 20°C incubation when cultured on wood blocks and wood chips. One strain of P. nordicum (not wood-associated) produced ochratoxin A when cultured on wood chips.     

The O:34-antigen lipopolysaccharide as an adhesin in Aeromonas hydrophila

Abstract We compared the ability of different Aeromonas hydrophila strains from serogroup O:34 grown at different temperatures to adhere to Hep-2 cells. We found a high level of adhesion when the strains were grown at 20 °C but not when they were grown at 37 °C. We previously described that these strains were able to form the O-antigen lipopolysaccharide when they grow at low temperature but not at high temperature. We also obtained by transposon mutagenesis mutants only devoid of the O-antigen lipopolysaccharide ( rfb mutants), and they showed significantly lower levels of adhesion to Hep-2 cells than the smooth strains. All these results prompted us to conclude that the O-antigen LPS, in these strains, is an important adhesin.     

Effect of various gas atmospheres on the growth and survival of Campylobacter jejuni on beef

Pieces of fresh beef were inoculated with three strains of Campylobacter jejuni . The meat was then allocated to three treatments: (a) vacuum packaged, (b) packaged in an atmosphere of 20% CO₂+ 80% N₂, and (c) packaged into sterile Petri dishes in anaerobic cultivation boxes, which were filled with a gas mixture of 5% O₂+ 10% CO₂+ 85% N₂. The packaging material in the first two treatments was PA 80/PE 100–PE 100/PA 80/PE 100. The survival of Campylobacter cells was followed at 37°C, 20°C and 4°C for 48 h, 4 days and 25 days, respectively. At 37°C the counts of two Campylobacter strains increased in each package treatment for 48 h. At 20°C and at 4°C the counts of the same two strains decreased by 1 to 2 log units and 0.5 to 1 log unit, respectively, during storage. The survival of the two strains was about the same in all package treatments. The third strain was the most sensitive of the strains studied. At 37°C its numbers increased only in the optimal gas atmosphere; at 20°C the strain was not detectable after 24 to 48 h storage and at 4°C after 4 days storage. The aerobic plate counts were determined for all samples at the same time as Campylobacter counts. The high indigenous bacterial numbers of the meat samples did not appear to have a great effect on the survival or growth of campylobacters.     

Homeostatic regulation of acetazolamide-sensitive esterase activity in the bluegill sunfish, Lepomis macrochirus, following acute cold shock

Acetazolamide-sensitive esterase activity was elevated in branchial homogenates of control juvenile bluegill sunfish, Lepomis macrochirus , acclimated at 20° C but decreased rapidly within 9 h following an acute hypothermal shock to 8° C. After 2 weeks at 8° C, shocked-fish enzyme activity was similar to control fish. At 20° C acclimation temperature, specific activity of bluegills was similar in swimbladder, liver, kidney, gill, spleen, and gonad homogenates and was significantly higher (α=0.05 level) in whole blood homogenates. The pH optima for enzymes extracted from fish acclimated at 20° and 8° C were 7.29 and 8.00, respectively. Polyacrylamide gel electrophoresis (PAGE) demonstrated two distinct forms of acetazolamide-sensitive esterase activity present in both 20° and 8° C acclimated fish. Specific activity for homogenates from both 20° and 8° C acclimated fish differed significantly when assayed at 20° C, suggesting both qualitative and quantitative changes in acetazolamide-sensitive esterase. It is postulated that relatively rapid alterations in esterase activity promote survival in bluegill following acute cold shock through the central role of enzymes in the regulation of plasma ion concentrations and acid/base equilibria.     

Chitinase profiles in mature carrot (Daucus carota) roots and purification and characterization of a novel isoform

The profile of chaitinases (EC 3.2.1.14) in mature carrot ( Daucus carota L. cv. Eagle) roots was studied. Multiple chitinase bands (8–10) were observed in native and sodium dodecylsulfate-denaturing polyacrylamide gels. The molecular masses of these chitinases were estimated to be from 20 000 to 40 000. One major chitinase was purified and found to be an acidic protein with pI at 4.3 and a molecular mass of 39 500. The optimum pH for enzymatic activity was around 5 and the optimum temperature was 25°C. The enzyme was stable at pHs below 8 and temperatures below 60°C. The protein did not have a chitin-binding domain, but showed some similarity to the amino acid composition of tobacco class I chitinase. The N-terminal amino acid sequence did not resemble any of the described classes of chitimases. This chitinase did not possess lysozyme activity and showed antifungal activity when tested against Trichoderma sp.     

Antibacterial activity of naringin derivatives against pathogenic strains

Aims: To study the antimicrobial activity of naringin (NAR), a flavonoid extracted from citrus industry waste, and NAR derivatives [naringenin (NGE), prunin and alkyl prunin esters] against pathogenic bacteria such as L. monocytogenes, E. coli O157:H7 and S. aureus. The relationship between the structure of the chemical compounds and their antagonistic effect was also analysed. Methods and Results: The agar dilution technique and direct contact assaying were applied. NGE, prunin and NAR showed no antimicrobial activity at a concentration of 0·25 mmol l^?1. Similarly, fatty acids with a chain length between C2 and C18 showed no antimicrobial activity at the same concentration. However, prunin‐6″‐O‐acyl esters presented high antibacterial activity, mainly against Gram‐positive strains. This activity increased with increasing chain length (up to 10–12 carbon atoms). Alkyl prunin esters with 10–12 carbon atoms diminished viability of L. monocytogenes by about 3 log orders and S. aureus by 6 log orders after 2 h of contact at 37°C and at a concentration of 0·25 mmol l^?1. The compounds examined were not effective against any of the Gram‐negative strains assayed, even at the highest concentration. Conclusions: Addition of sugars to the aglycone did not enhance its antimicrobial activity. Attachment of a saturated aliphatic chain with 10–12 carbon atoms to the A ring of the flavonoid (or to sugars attached to this ring), seems to be the most promising modification. In conclusion, alkyl prunin esters with a chain length of C10–C12 have promising features as antimicrobial agents because of their high antilisterial and antistaphylococcal activity. Significance and Impact of the Study: This study shows that it is possible to obtain NAR derivatives with important antimicrobial activity, especially against Gram‐positive pathogenic bacteria. It also provides guidelines on the structural modifications in similar molecules to enhance the antimicrobial activity.     

Antagonistic Action of the Bacterium Bacillus Licheniformis M-4 Toward the Amoeba Naegleria Fowleri

ABSTRACT. Free-living amoebae belonging to the species Naegleria fowleri are known to be the etiological agents for a form of fulminant meningoencephalitis that is generally fatal (primary amoebic meningoencephalitis). In a broad bacterial screening from soil and water we have isolated three strains (M-4, D-13 and A-12) belonging to the species Bacillus licheniformis that have remarkable amoebicidal activity against Naegleria sp. and also against different Gram-positive and Gram-negative bacteria. Physical-chemical characteristics, partial purification and biological activities of a substance produced by the M-4 strain have been investigated. This substance (m-4) is stable at high temperature (up to 100°C) and extremes of pH (2.5-9.5) and also at -20°C for months. Its production is greatly influenced by oxygenation of the cultures and is probably related to the sporulation process of the bacterium. Scanning electron microscope observations reveal that amoebae are lysed after a few minutes contact with m-4.     

Effect of temperature on the vegetative growth of type and field strains of Bacillus cereus

Growth of eight selected potentially pathogenic strains of Bacillus cereus was evaluated in a rich medium at different temperatures. No strain grew at 50°C; maximal growth-permissive temperatures were in the range 46–50°C for six strains and under 46°C for one strain. Faster growth occurred at 42°C. Growth may be delayed at 20°C, where the lag phase can reach 7 h. Furthermore at 20°C, cells generally show an aggregation immediately in the early exponential stage, except for two strains. Owing to this aggregation, growth is more difficult to estimate by turbidimetry at lower temperatures. These data describe the behaviour of type and field strains between 50° and 20°C and can help the prediction of shelf-life of potentially contaminated products.