Isolation and characterization of moderately halophilic bacteria from fully cured salted cod (bachalao)

A total of 128 Strains of moderately halophilic bacteria were isolated from wet, dry and desalted bachalao (salted codfish) as well as from fresh cod and curing salt. The viable count of these bacteria in fully cured wet and dry bachalao ranged from 10³ to 10⁷ per g. All strains were characterized with 40 phenotypic tests and clustered using the S _SM coefficient and UPGMA linking analysis. The strains clustered into five phena at 75% similarity, with 77 strains in phenon A and 37 in phenon E. Two main colony types, smooth and rough, were observed and correspond to phena A, B and C on one hand and phenon E on the other. These two types seem to represent the dominating bacterial flora in fully cured, wet and dry bachalao, respectively. Representative strains of the smooth colony type were characterized further and found to grow well in 0.1–4.5 mol I⁻¹ NaCl and at 15–37°C. They have not, at present, been assigned to any known bacterial species.     

Evaluation of the Growth of Salmonellae in Rappaport's Broth and in Muller-Kauffmann's Tetrathionate Broth

S ummary . The behaviour of 70 strains of salmonellae belonging to 44 serotypes in Rappaport's broth and in Muller-Kauffmann's tetrathionate broth was examined. With an inoculum of 5–25 cells, 5 strains did not grow in Rappaport's medium, 2 multiplied slowly and 63 grew strongly in 24 h. With an inoculum of 100–500 organisms all but one strain grew readily in 24 h. In Muller–Kauffmann's tetrathionate broth inoculated with pure cultures of salmonellae, growth of many strains was markedly inhibited, in the absence of added faeces, at 37° and 43°. This inhibition was more severe with light inocula at 43°. The addition of 0.05% (w/v) of salmonella-free human faeces to Muller–Kauffmann's tetrathionate broth, did not stimulate growth of salmonellae. In contrast, the addition of 5% (w/v) of human stools to this medium resulted in a heavy growth of the added salmonellae, especially at 43°.     

Minimum growth temperatures of Listeria monocytogenes and non-haemolytic listeria

Minimum growth temperatures and those of decreased growth were determined for 100 strains of listerias. The ability of 78 strains of Listeria monocytogenes isolated from animals and 22 non-haemolytic strains to grow at low temperatures was studied, using a flooding technique, in a plate-type continuous temperature gradient incubator at temperatures between –1.6 and 14.5.C. The mean minimum temperature for L. monocytogenes was +1.1 0.3.C. The growth of non-haemolytic listerias was unobservable at +1.7 0.5.C. The L. monocytogenes strains grew at about 0.6°C lower than the non-pathogenic strains. No differences in growth temperatures were observed among L. monocytogenes strains isolated from different sources. The serovars with the OI antigen grew at lower temperatures (+1.0.3°C) than the other common serovar 4b (+1.3 0.4°C).
The results indicate that L. monocytogenes grows better than non-haemolytic strains under cold conditions. The possible role of haemolysins as growth factors is also discussed.     

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.     

Numerical taxonomic study of thermophilic Bacillus isolated from three geographically separated deep-sea hydrothermal vents

Abstract: A numerical taxonomic study has been carried out with 80 strains, newly isolated, from three geographically separated deep-sea hydrothermal vents (Mid-Atlantic Ridge, Guaymas Basin and Lau Basin) and eleven thermophilic reference strains representing 11 Bacillus species. The deep-sea isolates were all halotolerant spore-forming rods and grew aerobically above 65°C. Results from unweighted average linkage cluster analysis of a similarity matrix derived from the simple matching coefficient, showed formation of nine major phena, which were defined at the 83% similarity level or above. Seven phena were composed exclusively of strains isolated from the same site (4 from Mid-Atlantic Ridge, 1 from Guaymas Basin and 2 from Lau Basin). The majority of the Lau Basin isolates clustered with 6 of the reference strains in one phenon, while isolates from Mid-Atlantic Ridge and Guaymas Basin were found separated from this phenon at the 69% similarity level. The other reference strains showed less than 69% similarity with the deep-sea isolates.     

Heat resistance of different serovars of Listeria monocytogenes

Twelve Listeria monocytogenes strains representing seven serovars were heat-treated in physiological saline by a glass capillary tube method. Five strains were treated at 58°, 60° and 62°C, three at 60°, 62° and 64°C and four at 60°C. Heat-treated bacteria were recovered on blood agar in two ways: (1) incubation at 37°C for 7 d; and (2) preincubation at 4°C for 5 d, followed by incubation at 37°C for 7 d. D and z values were determined. Better average recovery and higher D values were obtained when the preincubation procedure was used. The final evaluations of the heat resistance properties of the strains were therefore based on values for preincubated samples. D values recorded at 58°, 60°, 62° and 64°C for preincubated samples were 1.7–3.4, 0.72–3.1, 0.30–1.3 and 0.33–0.68 min, respectively. z values determined were 5.2–6.9°C. D values were compared statistically. Significant differences in heat resistance were noted both between serovars and between strains belonging to the same serovar.     

Partial purification and characterization of phospholipase C from Yersinia enterocolitica

About 34% of the strains of Yersinia enterocolitica isolated from raw milk were found to produce lecithinase. A selected strain produced phospholipase C at 22°C and 37°C; production was optimum at 37°C in the stationary phase (14–16 h). A decrease in phospholipase C activity at various storage temperatures (—5°C, 4°C, 37°C) was also observed, although the enzyme was active over a wide range of temperature (5–65°C) and pH (3mD5–7mD5). The phospholipase C was partially purified by ammonium sulphate precipitation and Sephadex column chromatography, and characterized.     

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.     

Thermophilic denitrifying bacteria: A survey of hot springs in Southwestern Iceland

Abstract Samples of water, sediment and bacterial mat from hot springs in Grændalur and Hveragerdi areas in southwestern Iceland were screened at 70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing nitrate or N₂O as the terminal oxidant. The springs ranged in temperature from 65–100°C and included both neutral (pH 7–8.5) and acidic (pH 2.5–4) types. Nitrate reducing bacteria (nitrate → nitrite) and denitrifiers (nitrate → N₂) were found that grew at 70°C but not at 80°C in nutrient media at pH 8. Samples from neutral springs that were cultured at pH 8 failed to yield a chemolithotrophic, sulfur-oxidizing and nitrate-reducing bacterium, and samples from acidic springs that were cultured at pH 3.5 seemed entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of growth solely by N₂O respiration. The denitrifiers appeared to be Bacillus . Two such Bacillus strains were examined in pure culture and found to exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosa , and one other strain of thermophilic Bacillus . The phenotype is characterized by the ability to grow by reduction of nitrate to N₂ with N₂O as an intermediate but a virtual inability to reduce N₂O when N₂O was the sole oxidant.     

Isolation and properties of psychrotrophic and psychrophilic, pectolytic strains of Clostridium

Fourteen strains of pectolytic clostridia have been isolated that were capable of growth at 5–10°C in 7 d; two strains were psychrophiles and failed to grow at 20°C in 14 d and the remainder were psychrotrophs. The bacteria formed pectate lyase enzymes and were capable of degrading potato tissue; they are therefore a potential cause of soft rot of potatoes stored at low temperatures. Doubling times for representative strains were 15–19 h at 10°C and 21–53 h at 5°C. Twelve strains were classified with Group I Clostridium species and two strains with Group II. In the case of one strain the mature spores were not released from the sporangium. Electron microscopy of ultrathin sections of this strain showed the presence of disorganized lamellar structures associated with the spore coat.     

Thermophilic denitrifying bacteria: A survey of hot springs in Southwestern Iceland

Abstract Samples of water, sediment and bacterial mat from hot springs in Grændalur and Hveragerdi areas in southwestern Iceland were screened at 70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing nitrate or N₂O as the terminal oxidant. The s springs ranged in temperature from 65–100°C and included both neutral (pH 7–8.5) and acidic (pH 2.5–4) types. Nitrate reducing bacteria (nitrate → nitrite) and denitrifiers (nitrate → N₂) were found that grew at 70°C but not at 80°C in nutrient media at pH 8. Samples from neutral springs that were cultured at pH 8 failed to yield a chemolithotrophic, sulfur-oxidizing and nitrate-reducing bacterium, and samples from acidic springs that were cultured at pH 3.5 seemed entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of growth solely by N₂O respiration. The denitrifiers appeared to be Bacillus . Two such Bacillus strains were examined in pure culture and found to exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosa , and one other strain of thermophilic Bacillus . The phenotype is characterized by the ability to grow by reduction of nitrate to N₂ with N₂O as an intermediate but a virtual inability to reduce N₂O when N₂O was the sole oxidant.     

Growth rate and physiology of Yersinia enterocolitica; influence of temperature and presence of the virulence plasmid

The effect of temperature on the growth rate, protein pattern and fatty acid composition of Yersinia enterocolitica strain W22703 pYV⁺, its plasmidless isogenic derivative W22703 pYV^- and four recent field isolates was examined.
The growth rate was clearly influenced by presence or absence of the virulence plasmid: pYV^- strains grew consistently faster than pYV⁺ strains. This difference in growth rate was high at 30–35°C, moderate at 1–10°C and 25°C, but hardly significant at 15–20°C.
Increasing the growth temperature above 25°C resulted in the induction of the 220 kDa virulence plasmid-encoded Yop1 protein. In the 1–20°C range no obvious temperature- or plasmid-related differences in protein patterns could be detected.
The fatty acid composition showed a clear temperature-dependent change: with all strains the degree of saturation was low at 1°C and gradually increased with raising temperatures. All strains had similar fatty acid patterns, except one of the field isolates which showed aberrant C16 : 1 and cyclic fatty acid contents in the 5–25°C and 15°C ranges respectively. With strain W22703, the presence or absence of the virulence plasmid did not significantly alter the fatty acid pattern.     

Survival and growth of Cronobacter species (Enterobacter sakazakii) in wheat-based infant follow-on formulas

Aims:  To determine the survival and growth characteristics of Cronobacter species ( Enterobacter sakazakii ) in infant wheat-based formulas reconstituted with water, milk, grape juice or apple juice during storage.
Methods and Results:  Infant wheat-based formulas were reconstituted with water, ultra high temperature milk, pasteurized grape or apple juices. The reconstituted formulas were inoculated with Cronobacter sakazakii and Cronobacter muytjensii and stored at 4, 25 or 37°C for up to 24 h. At 25 and 37°C, Cronobacter grew more (>5 log₁₀) in formulas reconstituted with water or milk than those prepared with grape or apple juices ( c. 2–3 log₁₀). The organism persisted, but did not grow in any formulas stored at 4°C. Formulas reconstituted with water and milk decreased from pH 6·0 to 4·8–5·0 after 24 h, whereas the pH of the formulas reconstituted with fruit juices remained at their initial pH values, c. pH 4·8–5·0.
Conclusions:  Cronobacter sakazakii and C. muytjensii can grow in reconstituted wheat-based formulas. If not immediately consumed, these formulas should be stored at refrigeration temperatures to reduce the risk of infant infection.
Significance and Impact of the Study:  The results of this study will be of use to regulatory agencies and infant formula producers to recommend storage conditions that reduce the growth of Cronobacter in infant wheat-based formulas.     

Psychrotrophic Clostridia

Of 287 strains of clostridia of 23 different species 96 grew well at 6°, 77 of which were Clostridium carnofoetidum var. amylolyticum and 16 were Cl. perfringens.
The water of polluted wells seems to be the normal habitat of Cl. carnofoetidum var. amylolyticum . This species is both proteolytic and saccharolytic and is not pathogenic for mice or guinea pigs. Spores survive at 80° for 40 min. Mg is essential to obtain sporulation of the strains in artificial environments.
Growth temperatures vary between 6 and 37° with an optimum at 30°. At 6° strains untrained to grow at this temperature grow in about 5 days in Rosenow medium. Trained stains have a lag phase of 24 h. Growth curves show a longer logarithmic phase at 6° than at 30°. At the end of this phase the bacterial count is the same at both temperatures. Cl. carnofoetidum var. amylolyticum is, therefore, a true psychrotrophic species.
Glucose and pyruvate breakdown studied by Warburg's method confirms the slowing down of metabolism at 6°. A study of amino acid degradation indicates that serine is the substance most rapidly attacked at 37°, but at 6° the activity against serine disappears completely.
In Rosenow medium at the first inoculation 8 strains of Cl. perfringens out of 24 failed to grow at 6° after 30 days. They were all enterotoxic types. Among the other 16 strains none survived beyond the tenth transfer at 6° and it was not possible to train any strain to grow at this temperature. An examination of the growth curves shows that they are not truly psychrotrophic.     

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.     

Effect of temperature on sulphate reduction, growth rate and growth yield in five psychrophilic sulphate-reducing bacteria from Arctic sediments

Five psychrophilic sulphate-reducing bacteria (strains ASv26, LSv21, PSv29, LSv54 and LSv514) isolated from Arctic sediments were examined for their adaptation to permanently low temperatures. All strains grew at −1.8°C, the freezing point of sea water, but their optimum temperature for growth ( T _opt) were 7°C (PSv29), 10°C (ASv26, LSv54) and 18°C (LSv21, LSv514). Although T _opt was considerably above the in situ temperatures of their habitats (−1.7°C and 2.6°C), relative growth rates were still high at 0°C, accounting for 25–41% of those at T _opt. Short-term incubations of exponentially growing cultures showed that the highest sulphate reduction rates occurred 2–9°C above T _opt. In contrast to growth and sulphate reduction rates, growth yields of strains ASv26, LSv54 and PSv29 were almost constant between −1.8°C and T _opt. For strains LSv21 and LSv514, however, growth yields were highest at the lowest temperatures, around 0°C. The results indicate that psychrophilic sulphate-reducing bacteria are specially adapted to permanently low temperatures by high relative growth rates and high growth yields at in situ conditions.     

SOME BIOCHEMICAL CHARACTERISTICS OF FRESHLY ISOLATED STRAINS OF THE GENUS SERRATIA

SUMMARY: Many of 110 strains of Serratia , isolated from soil, water, milk and dairy equipment, were biochemically closely related to the coli-aerogenes bacteria. Acid and gas was formed from glucose in 14 days at 30° by 53% and from lactose and MacConkey's broth by about 40%. All except one strain gave——++ IMViC reactions.
An inverse relationship was observed between depth of pigmentation and carbohydrate fermentation. Complete loss of pigment in mutant strains was not uncommon, and was associated with loss of proteolytic properties and increase of saccharolytic activity.
The majority of the strains had psychrophilic characteristics: 75% grew at 3–5°. Most strains showed moderate growth at 37°, but only 7 formed red pigment at that temperature.
All strains resembled Serratia marcescens in morphology, containing minute coccoid rods smaller than those of coli-aerogenes bacteria.     

The influence of pH and temperature on initiation of growth of Salmonella spp.

The ability of 13 strains of Salmonella , representing 12 serotypes, to grow in a tryptone-yeast extract-glucose medium, acidified with HC1 to pH values between 3.80 and 5.60 at intervals of 0.20 units, has been investigated. During incubation at 30°C, growth occurred at minimum pH values of 3.8–4.0 in 1–3 d. At 20°C, growth occurred at minimum pH values of 3.8–4.2 in 3–5 d. In tests incubated at 10°C, growth occurred at minimum pH values of 4.4–4.8 in between 10 and 19 d.     

Heat-resistant Bacteria in Pasteurized Whole Egg

Samples of egg melange taken from an egg packing station contained an average of 7·3 x 10⁴ organisms/ml which survived laboratory pasteurization at 65°C for 3 min. Many of the organisms surviving pasteurization were found to be coryneform bacteria related to Microbacterium lacticum which could be differentiated into several groups. The remainder were a miscellaneous collection of unidentified cocci and coccobacilli and some Bacillus spp. The coryneform bacteria were shown to be the most heat-resistant isolates with negligible loss of viability after 60 min at 65°C, At least two of the representative strains were very heat-resistant, 0·01% surviving 20 and 38 min at 80°C in phosphate buffer at pH 7·1. Growth tests showed that none of the isolates grew at 5°C after 10 d incubation but those capable of growing most rapidly at 10° and 15°C were also the most heat-resistant. Such strains had a doubling time at 15°C of between 6 and 8 h in whole egg. Freezing the coryneform bacteria in liquid whole egg at –18°C had negligible effect on viability or heat-resistance at 65°C.     

Morphological dormancy in seeds of the autumn-germinating shrub Lonicera caerulea var. emphyllocalyx (Caprifoliaceae)

To better understand the germination ecophysiology of the genus Lonicera , the dormancy class, temperature requirements for embryo growth and radicle emergence and phenology of seedling emergence were determined for Lonicera caerulea var. emphyllocalyx . At maturity, seeds have an underdeveloped embryo (approximately 28% of the length of full-grown embryos). Embryos in fresh seeds grew to full length at 15, 20, 20/10 and 25/15°C within 3 weeks, but failed to grow at ≤ 10°C and at 30°C. Radicles emerged from 86–100% of freshly matured seeds in light at 15, 20, 20/10 and 25/15°C within 28 days, but failed to emerge at 10°C. Radicles emerged equally well in a 12 h photoperiod and in continuous darkness at 25/15°C. Rapid embryo growth and germination over a range of conditions indicate that seeds of this taxon have morphological dormancy (MD); this is the first report of MD in a species of Lonicera . Seeds are dispersed in summer, at which time high temperatures promote embryo growth. Embryos grow to the critical length for germination in approximately 1 month; the peak of seedling emergence occurs in early autumn. Radicles emerged within 2 months from 98% of seeds buried at soil depths of 2 cm and 10 cm in the field in August in Sapporo, Japan; thus, seeds have no potential to form a persistent soil seed bank. However, seeds sown too late in autumn for embryos to grow remained viable and germinated the following summer when temperatures were high enough to promote embryo growth.