Tracheobronchial washings from seven vertebrate species as growth media for the four species of Bordetella

Abstract The four species of Bordetella differed in their ability to grow at 37°C in membrane-filtered tracheobronchial washings (TBW) from seven vertebrate species, including their natural hosts. From washed inocula of approximately 2×10³ colony-forming units per ml (cfu ml⁻¹), Bordetella bronchiseptica and B. avium grew much better than the other two bordetellae and yielded stationary-phase cultures containing 10⁸−10⁹ cfu ml⁻¹ in most of the TBW samples. These counts were only moderately lower than those attained in CL medium which contains about a 450-times higher concentration of amino acids. B. bronchiseptica and B. avium also grew to a limited extent in phosphate-buffered saline without nutrient supplements. B. parapertussis grew in TBW from man, sheep, rabbit, mouse and chicken, but not in TBW from a dog and a horse or in PBS. B. pertussis grew well in CL medium, but not in PBS or in any of 13 samples of TBW from the seven vertebrate species, which included three samples of lung lavage fluid from human patients. Analysis of the TBW samples for known Bordetella nutrients revealed concentrations of amino acids and nicotinic acid averaging 0.35 mM and 0.56 μg ml⁻ respectively.     

Effect of chill and freezing temperatures on survival of Vibrio parahaemolyticus inoculated in homogenates of oyster meat

Survival of Vibrio parahaemolyticus was determined in oyster meat homogenates at various temperatures. (4°C, 0°C, -18°C and -24°C) and bacterial levels (10², 10⁴, 10⁵ and 10⁷ ml^-1). In all cases, the numbers of V. parahaemolyticus were a logarithmic function of log time. This study indicates that high numbers of V. parahaemolyticus can be inactivated at low temperatures. The time of total inactivation depends on the initial number of micro-organisms and incubation temperature. It is possible to use this information to determine the storage time necessary to reduce V. parahaemolyticus hazards in fish.     

Scaling of oxygen consumption of Lake Magadi tilapia, a fish living at 37°C

Rates of oxygen consumption were measured in the geothermal, hot spring fish, Oreochromis alcalicus grahami by stopped flow respirometry. At 37° C, routine oxygen consumption followed the allometric relationship: V o₂=0.738 M ^0.75, where V o₂ is ml O₂ h ⁻¹ and M is body mass (g). This represents a routine metabolic rate for a 10 g fish at 37° C of 0.415 ml O₂ g⁻¹ h ⁻¹ (16.4 μmol O₂ g ⁻¹ h ⁻¹). Acutely increasing the temperature from 37 to 42° C significantly elevated the rate of O₂ consumption from 0.739 to 0.970 ml O₂ g ⁻¹ h ⁻¹ ( Q ₁₀=l.72). In the field, O. a. grahami was observed to be 'gulping' air from the surface of the water especially in hot springs that exceeded 40° C. O. a. grahami may utilize aerial respiration when O₂ requirements are high.     

The effect of procaine on the partitioning of plasmid pSC101

Abstract An examination of samples obtained from a commercial fish smoker, using seawater agar with incubation at 4°, 15° and 37°C for up to 28 days, revealed the presence of large bacterial populations in smoked fish. However, initially only low bacterial numbers, i.e., 2 × 10³/g, were present in the muscle of fresh, whole haddock ( Melanogrammus aeglefinus ). With filleting, there was a sudden increase in numbers to 9.2 × 10⁵/g. Yet immediately after smoking, the bacterial populations decreased (5 × 10⁵/g), followed by a gradual increase with storage (e.g., 2 × 10⁶/g after 24 h). Representative colonies were presumptively identified as Acinetobacter, Alcaligenes , coryneforms, Pseudomonas and Vibrio spp.     

Survival of Escherichia coli O157 : H7 in yoghurt during preparation and storage at 4°C

Cow's milk was inoculated with ca 10³ and 10⁷ cfu ml⁻¹ Escherichia coli O157 : H7. After fermentation at 42°C for 0–5 h, the yoghurt was stored at 4°C. Two kinds of yoghurt were used : traditional yoghurt (TY), made with Streptococcus thermophilus and Lactobacillus bulgaricus starter cultures, and 'bifido' yoghurt (BY), made with the two starter cultures plus Bifidobacterium bifidum . After 7 d E. coli O157 : H7 decreased from 3·52 to 2·72 log₁₀ cfu ml⁻¹ and from 7·08 to 5·32 log₁₀ cfu ml⁻¹ in TY, and from 3·49 to 2·73 log₁₀ cfu ml⁻¹ and from 7·38 to 5·41 log₁₀ cfu ml⁻¹ in BY. The pH values of yoghurt dropped from 6·6 to 4·5 and 4·4 in TY (for low and high pathogen inocula, respectively), and from 6·6 to 4·6 and 4·5 in BY (for low and high pathogen inocula, respectively).     

Efficacy of Beauveria bassiana (Bals.) Vuill. against the spruce bark beetle, Ips typographus L., in the laboratory under various conditions

Abstract:  In laboratory bioassays, the efficacy of the entomopathogenic fungus Beauveria bassiana against the spruce bark beetle, Ips typographus , was tested under various conditions. Four of the tested isolates and the commercial product Boverol^® caused 99–100% mortality when tested at a concentration of 1.0 × 10⁷ conidia/ml at 25°C. Using B. bassiana isolate 138 at a concentration of 1.0 × 10⁶, the median survival time (MST) was 6.1 d and significantly longer compared with the MST of 4.2 and 4.0 d at 1.0 × 10⁷ and 1.0 × 10⁸ conidia/ml, respectively. In the next experiment, the beetles were maintained on spruce bark, filter paper or artificial diet during the bioassay with Boverol^®, and significant differences in the MST of 3.6, 2.5 and 5.3 d, respectively, were noticed. The experiment with Boverol^® at different temperatures showed that the beetles lived significantly longer at 15°C (MST 8.7 d) than at 20, 25, 30 and 35°C. At 25°C, the beetles died most rapidly (MST 3.5 d). At different relative humidities (RH) of 40, 70 and 100%, nearly all beetles were dead after treatment with a suspension of Boverol^® at 1.0 × 10⁷ conidia/ml. At 40% RH, 49% of the untreated beetles died after 7 d. The best effects were achieved with the following bioassay: beetles were fed for three days on artificial diet, then dipped into a solution of 1.0 × 10⁷ conidia/ml and transferred on a piece of spruce bark in Petri dishes at 25°C and 70% RH.     

In Vitro Effect of Tetanus Toxin on GAB A Release from Rat Hippocampal Slices

Abstract Ca²⁺-dependent K⁺-stimulated γ-aminobutyric acid release from rat hippocampal slices was reduced about 30% by pre-incubation of the slices with 10⁴ mouse LD₅₀/ml tetanus toxin for 3 h at 37°C.     

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.     

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.     

A Comparison of Toxin Production by Clostridium botulinum Type E in Irradiated and Unirradiated Vacuum Packed Fish

S ummary . The rates of toxin production at 10° by inocula of 10⁵, 10⁴, 10³ and 10² spores of Clostridium botulinum type E in vacuum packed herring, cod and haddock were compared with that of equivalent numbers of spores surviving γ-irradiation at 0.3 Mrad. There was little difference between the rates of toxin production in unirradiated and irradiated fish. More toxin was produced in irradiated herring than in unirradiated, but in haddock the reverse was obtained. In cod about equal amounts of toxin were found without trypsinization; after trypsinization toxin levels were generally higher in irradiated samples.     

Temperature effects on the viable but non-culturable state of Vibrio vulnificus

Abstract The non-culturable state of Vibrio vulnificus , strain C7184, was studied in artificial seawater microcosms held at 5, 10, 15, 20, and 30°C. Plate counts were made on a non-selective medium, total cell counts were monitored by acridine orange epifluorescence, and direct viable counts (DVSs) by the method of Kogure et al. (Can J. Microbiol. 25, 415–420; 1986) and by the INT method. From an initial inoculum of 10⁷ cells/ml, V. vulnificus became non-culturable within 40 days at 5°C, although both indicators of viability revealed a viable population exceeding 10⁶ cells/ml. Cells at all higher temperatures remained culturable (at least 10⁴/ml) throughout the study. The non-culturable states of the opaque and translucent colony variants of V. vulnificus , as well as those of six other clinical and environmental strains of V. vulnificus , were examined at 5°C; all but one strain and both colony variants also became non-culturable within 40 days. In contrast, six other Vibrio spp. ( V. cholerae, V. mimicus, V. parahaemolyticus, V. natriegens, V. proteolyticus , and V. campbelli ) remained culturable at 5°C. Thus, entrance of V. vulnificus into the non-culturable state appears to be highly temperature dependent and, among the vibrios, this species may be especially sensitive to low temperature. The public health aspects of these findings are discussed.     

The development of a conductimetric assay for automated detection of metabolically active soft rot Erwinia spp. in potato tuber peel extracts

Four media were tested for their ability to detect the soft rot potato pathogens Erwinia chrysanthemi (Ech) and Erwinia carotovora ssp. atroseptica (Eca) in potato tubers by means of automated conductance measurements. The specificity of the conductimetric assays was determined by testing a set of different Erwinia spp. and potato-associated saprophytes, including the genera Pseudomonas, Bacillus, Enterobacter and Flavobacterium. All bacteria tested produced conductance responses in Special Peptone Yeast Extract, whereas in minimal medium with L-asparagine only Erwinia spp. and Pseudomonas spp. were able to generate large conductance responses. In minimal medium supplemented with glucose and trimethylamine- N -oxide only Enterobacteriaceae, Erwinia spp. included, generated conductance responses, while with pectate as sole carbon source only Erwinia spp. produced distinct conductance responses. The pectate medium proved to be particularly useful for specific automated conductimetric detection of Erwinia spp. in potato peel extracts. Within 48 h, the detection threshold of the conductimetric assay for Eca varied between 10² and 10³ cfu per ml peel extract at both incubation temperatures of 20° and 26°C. Ech was detected at concentrations of 10⁴–10⁵ or 10³–10⁴ cfu ml^-1 at 20° and 26°C, respectively. To eliminate 'false'-positive reactions in conductimetry caused by Erwinia carotovora ssp. carotovora , results of the conductance measurements have to be confirmed by other techniques, like serology or DNA assays.     

Improvement in the growth performance of white shrimp, Litopenaeus vannamei, by a protease-producing probiotic, Bacillus subtilis E20, from natto

Aims:  To isolate and identify a benefic bacterium, Bacillus subtilis E20, from natto (fermented soybeans), and incorporate it into shrimp feed to promote shrimp growth performance.
Methods and Results:  A protease-producing bacterium, E20, isolated from natto was identified as B. subtilis by an API 50 CHB kit and the 16S rDNA sequence. B. subtilis E20 was able to grow at a broad range of temperatures (10–50°C), pH values (5–10), and NaCl levels (0–9%). The best culture conditions for B. subtilis E20 to produce the protease were 40°C, a pH of 6–8 and 0% NaCl. No shrimp died after being injected with B. subtilis E20 [up to 10⁹ colony-forming units (CFU) per shrimp]. Bacillus subtilis E20 was incorporated in diets at the levels of 0 (control), 10⁶, 10⁷, and 10⁸ CFU kg⁻¹ for shrimp grow-out culture, and results showed that after feeding on B. subtilis E20-containing diets (10⁸ CFU kg⁻¹ of diet), shrimp had excellent growth performance and production compared to the control because protease activities in the digestive tract were improved by B. subtilis E20.
Conclusions:  Bacillus subtilis E20 isolated from natto is a great protease producer and is able to improve shrimp growth performance through increasing the digestibility of food.
Significance and Impact of the Study:  Results suggest that B. subtilis E20 is a potential candidate for use as a probiotic to improve shrimp growth performance, and consequently reduce feed costs.     

Purification and biochemical characterization of a membrane-bound [NiFe]-hydrogenase from a hydrogen-oxidizing, lithotrophic bacterium, Hydrogenophaga sp. AH-24

Membrane-bound [NiFe]-hydrogenase from Hydrogenophaga sp. AH-24 was purified to homogeneity. The molecular weight was estimated as 100±10 kDa, consisting of two different subunits (62 and 37 kDa). The optimal pH values for H₂ oxidation and evolution were 8.0 and 4.0, respectively, and the activity ratio (H₂ oxidation/H₂ evolution) was 1.61 × 10² at pH 7.0. The optimal temperature was 75 °C. The enzyme was quite stable under air atmosphere (the half-life of activity was c . 48 h at 4 °C), which should be important to function in the aerobic habitat of the strain. The enzyme showed high thermal stability under anaerobic conditions, which retained full activity for over 5 h at 50 °C. The activity increased up to 2.5-fold during incubation at 50 °C under H₂. Using methylene blue as an electron acceptor, the kinetic constants of the purified membrane-bound homogenase (MBH) were V _max=336 U mg⁻¹, k _cat=560 s⁻¹, and k _cat/ K _m=2.24 × 10⁷ M⁻¹ s⁻¹. The MBH exhibited prominent electron paramagnetic resonance signals originating from [3Fe–4S]⁺ and [4Fe–4S]⁺ clusters. On the other hand, signals originating from Ni of the active center were very weak, as observed in other oxygen-stable hydrogenases from aerobic H₂-oxidizing bacteria. This is the first report of catalytic and biochemical characterization of the respiratory MBH from Hydrogenophaga .     

Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis

The efficacy of high-temperature, short-time (HTST) pasteurization (72 °C/15 s) when low numbers (≤ 10³ cfu ml ⁻¹ ) of Mycobacterium paratuberculosis are present in milk was investigated. Raw cows' milk spiked with Myco. paratuberculosis (10³ cfu ml⁻¹, 10² cfu ml⁻¹, 10 cfu ml⁻¹, and 10 cfu 50 ml⁻¹) was subjected to HTST pasteurization using laboratory pasteurizing units. Ten bovine strains of Myco. paratuberculosis were tested in triplicate. Culture in BACTEC Middlebrook 12B radiometric medium detected acid-fast survivors in 14·8% and 10% of HTST-pasteurized milk samples at the 10³ and 10² cfu ml⁻¹ inoculum levels, respectively, whereas conventional culture on Herrold's egg yolk medium containing mycobactin J detected acid-fast survivors in only 3·7% and 6·7% of the same milk samples. IS900-based PCR confirmed that these acid-fast survivors were Myco. paratuberculosis . No viable Myco. paratuberculosis were isolated from HTST-pasteurized milk initially containing either 10 cfu ml⁻¹ or 10 cfu 50 ml⁻¹.     

The effect of temperature and mass on routine metabolism in Sarotherodon (Tilapia)mossambicus (Peters)

A reappraisal of oxygen uptake by Sarotherodon mossambicus was undertaken using a continuous flow respirometer. Measurements were obtained over the temperature range 16°C–37°C for fish weighing between 10 g and 150 g. Oxygen uptake was converted to energy equivalents ( Q _ox) using the value 13.68 J mg O₂^–1and the routine metabolic energy expenditure can be described by the equation E =0.0086 t ^{2 0783} M ^{0 652} where E is the energy requirement for routine metabolism expressed in J h^-1, t the temperature in °C and M the mass in g.     

Interaction between the effects of bacteria and dry storage on the opening and water relations of cut rose flowers

W.G. VAN DOORN AND K. D'HONT. 1994. Flowering stems of four rose cultivars (Sonia, Madelon, Jacaranda and Frisco) were placed in aqueous suspensions of bacteria at 10⁴ and 10⁸ colony-forming units (cfu) ml^-1 for 24 h at 5C, then stored dry or held in water for 24 h at 8C and subsequently placed in vase-water at 20C. The effects of these treatments on vase-water uptake were similar to the effects on flower opening. In Sonia and Madelon roses flower opening was negatively affected both by 10⁸ cfu ml^-1 of bacteria and by dry storage. No effect was found at 10⁴ cfu ml^-1, but this concentration had a detrimental effect on flower opening when combined with dry storage. Although flower development in Jacaranda roses was not affected by the bacteria treatments it was inhibited by dry storage. This inhibition was progressively greater when the stems had previously been pulse-treated with a larger number of bacteria. Flower opening in Frisco roses was not affected by even the highest concentration of bacteria, nor by the period of dry storage. It is concluded that placing flowers in water containing bacteria (up to 10⁸ cfu ml^-1) may not always have a negative effect on flower development in cut rose flowers but, together with the effects of dry storage, the presence of even a low number of exogenous bacteria (10⁴ cfu ml^-1) inhibits the development in several cultivars. Such bacterial counts are nearly always found in samples of water used for standing roses during distribution to the consumers.     

Allocation of carbon to growth, storage and respiration in the vegetative barley plant

Abstract. It is proposed that the growing plant can be divided into three compartments with reference to carbon: soluble, storage and structural. Experiments carried out at 10, 15, 20 and 30°C in the light followed changes in size of these compartments in barley plants 10–24 days old. The redistribution of ^I4C photo-assimilated by 10 day old plants was monitored simultaneously. The soluble and storage compartments are a higher percentage of plant weight at lower temperatures, and are turned over rapidly at all temperatures; they form the source of respired ¹⁴C. About 30% of the ¹⁴C fixed enters structural material; in the first 24 h after labelling, for each unit of ¹⁴C entering the structural compartment, between 0–9 (at 15°C) and 3.2 (at 30°C) units of ¹⁴C are lost by respiration. At 15°C in the dark, respiratory loss of ¹⁴C is initially from soluble and storage compartments; thereafter respiration of ^I4C occurs at the expense of structural material.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5·2 with HCl and incubated at 30°C, inocula containing < 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give > 10⁸ bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10–20 weeks. Citric acid concentrations of 10–50 mmol/l at pH 5·2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10⁶. The citric acid also reduced the concentration of free Ca²⁺ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5·2 could be prevented by the addition of Ca²⁺ or Mg²⁺ and greatly reduced by Fe²⁺ and Mn²⁺. The addition of Ca²⁺, but not of the remaining divalent metal ions, restored the concentration of free Ca²⁺ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca²⁺. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5·2. The effect of citric acid and Ca²⁺ at pH 5·2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.     

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.