Study of substances released by ultrasonic treatment from Bacillus stearothermophilus spores

P. PALACIOS. J. BURGOS, L. HOZ, B. SANZ AND J.A. ORDÓÑEZ. 1991. Spore heat resistance is reduced when combined ultrasonic and heat treatments are applied. To explain this phenomenon the substances released from Bacillus stearothermophilus spores to the surrounding aqueous medium by the ultrasonic treatment (20 kHz, 120 W, 12°C, 30 min) were studied. Calcium, dipicolinic acid and a glycopeptide of 7 kDa molecular weight were detected in the ultrasonicated medium. Fatty acids, acyl glycerols and glycolipids (but no phospholipids) were also released. The decrease of heat resistance induced by the ultrasonic treatment was attributed to the release of low molecular weight substances from the spore protoplast with consequent modification of its hydration state.     

Effect of heat and ultrasonic waves on the survival of two strains of Bacillus subtilis

The combined effect of ultrasonic (20 KHz, 150 W) and heat treatment on the survival of two strains of Bacillus subtilis in three suspending media (distilled water, glycerol and milk) has been studied. When spores suspended in water or milk were subjected to ultrasonic waves before heat treatments a little or no decrease of the heat resistance was observed. However, both sporicidal agents applied simultaneously (thermo-ultrasonication) decreased by 63% (B. subtilis, var. niger-40) and 74% (B. subtilis ATCC 6051) the decimal reduction times for the heat treatment when the spores were suspended in glycerol and by 79% and 40%, respectively when suspended in milk. The thermo-ultrasonication of spores in water markedly reduced the heat resistance of them (between 99.9% and 70%) in the range 70-95 degrees C but the effect of the thermo-ultrasonication significantly diminished as the temperature of the treatment was approached to the boiling point of the water.     

Rapid labelling of bacteria with [14C]palmitic acid and its application in an aggregation assay

Abstract A rapid and simple procedure for labelling bacterial cells based on binding of [¹⁴C]palmitic acid to the bacterial surface is described. The method was found convenient for both Gram-positive and Gram-negative bacteria such as staphylococci, streptococci and Salmonella . Some factors affecting the binding of [¹⁴C]palmitic acid to the surface of streptococci were examined. Treatment of bacteria with heat, trypsin or β-galactosidase had no effect on labelling. The binding was relatively independent of ionic strength in the range 0.1–1 M NaCl, but was dependent on pH and presence of detergents. The [¹⁴C]palmitic acid labelling method was tested in studies of aggregation of oral streptococci. The aggregation assay was sensitive and very reproducible.     

THE RATES OF GROWTH OF SOME THERMODURIC BACTERIA IN PURE CULTURE AND THEIR EFFECTS ON TESTS FOR THE KEEPING QUALITY OF MILK

SUMMARY: The growth rates of eleven representative thermoduric bacteria, comprising 3 aerobic spore formers, 3 streptococci, 1 Corynebacterium lacticum and 4 micrococci, have been determined in glucose broth and sterile pasteurized milk at 37·5°, 26° and 15°. The spore formers and streptococci were generally not affected by the presence of inhibitory factors in pasteurized milk. When multiplication of micrococci and C. lacticum occurred in milk this was only after a lag period. One micrococcus showed an unusual series of growth phases in glucose broth at 37·5°, possibly due to the appearance of mutants or to adaptation of the organism to growth at that temperature. This was not observed in pasteurized milk. C. lacticum died off when incubated in glucose broth at 37·5°.
None of the keeping quality tests was more effective than any other in detecting these organisms in milk. The micrococci and C. lacticum had little effect on the keeping quality of pasteurized milk within the period of 'commercial life'. Some of the spore formers and streptococci showed marked differences in the end-points with the clot-on-boiling and the alcohol precipitation tests.     

Survey of the plasmid content of group A streptococci

Abstract Examination of 70 M-prototype group A streptococci showed small plasmids (2.0–2.5 MDa) to be present in strains representative of M-types 28, 57, 61, 63, 64 and 69. Identical results were obtained from M _r and restriction endonuclease analyses of a 2.2-MDa plasmid (pDN691) found in the M-type 69 strain and similar plasmids in the M57 prototype and two other independently isolated M-type 57 strains. In all four strains the presence of plasmid correlated with the production of bacteriocin-like inhibitory activity identifiable as P type 614. Similar analysis revealed a possible relationship between a 2.5-MDa plasmid in the prototype M61 and M64 strains and the production of P-type 216 inhibitory activity. A survey of 56 group A streptococci recovered in association with either rheumatic fever or nephritis failed to demonstrate plasmid DNA with the exception of 2.2-MDa plasmids in four nephritis-associated M-type 57 isolates.     

Effect of chilling, heat shock, and vigor on the growth of cucumber (Cucumis sativus) radicles

Chilling at 2.5°C reduced the subsequent growth of cucumber ( Cucumis sativus L.) radicles at 25°C. The reduction in radicle growth was linear for 1–3 days of chilling at ≈10% per day of treatment, but then it increased in a non-linear pattern until subsequent radicle growth was all but eliminated by 6 days of chilling. A heat shock of 40°C for 4–12 min increased chilling tolerance such that 4 days of chilling caused only a 36% decrease in radicle growth, compared to 66% for seedlings not heat shocked. Heat shocks were only able to protect that part of radicle growth that was in excess of the linear decrease in radicle growth projected from 0–3 days. There appear to be two effects of chilling on radicle growth. The first inhibition of subsequent growth was linear and was not affected by heat shocks. The second inhibition was much more severe; it appeared after 3 days of chilling and could be prevented by heat shock. Seeds classified with different levels of vigor (i.e., different initial rates of growth) did not respond significantly different to chilling stresses following heat-shock treatments.     

STUDIES OF THE BACTERIAL CONTENT OF WASHED MILK CANS

SUMMARY: Colony counts at 30° were greater than 10 times those at 37° in a high proportion of rinses of washed milk cans, the difference being most marked in those containing milk scale, where 58% of the colony counts at 30° exceeded 10⁶/can. A high proportion of the microflora was composed of thermoduric bacteria. Of 895 cultures from the milk scale, 33% were micrococci, 28% corynebacteria, 22% streptococci, and 9% were Gram-negative rods. Though aerobic sporing rods constituted only 5% of the microflora of the milk scale, they were present in large numbers and unsatisfactorily washed cans probably constitute one of the main sources of these organisms in milk.     

Effect of Bacillus thuringiensis on Habrobracon hebetor during combined biological control of Plodia interpunctella

Abstract  The suitability of combining microbial pesticides and an insect parasitoid for pest management of stored cereal in China was evaluated using laboratory assays. For this purpose, interactions between Bacillus thuringiensis ( Bt ), Bt -intoxicated host larvae and the parasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae) were tested against Plodia interpunctella Hübner (Lepidoptera: Pyralidae) . Bt or H. hebetor alone caused 41.67% and 35.35% P. interpunctella larval mortality respectively. The Bt –parasitoid combined treatment significantly increased mortality of P. interpunctella (86%). Progeny development of H. hebetor was dependent upon its susceptibility to Bt . Fewer parasitoids emerged from Bt –parasitoid combined treatment than in non- Bt treatments. However, since Bt did not prevent parasitoid development, a combined treatment with Bt and parasitoid release could produce better protection against P. interpunctella than either treatments when used singly, because their lethal effects were additive to each other.     

Cell surface protein receptors in oral streptococci

Abstract Streptococci have a vast repertoire of adherence properties which include binding to human tissue components, epithelial cells and to other bacterial cells. These interactions are determined by the expression of cell-surface receptors some of which are species-specific. In the oral streptococci, two families of surface protein receptors with highly conserved amino acid sequences have been identified. The antigen I/II family of polypeptides are wall-associated high molecular mass proteins (158–166 kDa) with several binding functions that may be attributed to different domains of the receptor molecules. The LraI family of polypeptides are surface-associated lipoproteins (32–33 kDa) involved in adherence of streptococci to salivary glycoprotein pellicle and to oral Actinomyces . A region of amino acid sequence similarity is evident amongst members of the two protein families in Streptococcus gordonii . Ligand-binding specificities of these receptor polypeptides may account for species-specific adherence and site-directed colonization of streptococci within the human oral cavity.     

Nucleic acid hybridization and cell wall composition studies of pyogenic streptococci

Abstract DNA-rRNA and DNA-DNA hybridization studies indicate that the classical pyogenic streptococci can be divided into five homology clusters. Based on these studies the term pyogenic streptococci should be confined to the first cluster consisting of serological groups A, A-variant, C, G ('large' colony, type II) and L.
Streptococci of serological groups B and M form the second cluster. The third cluster is composed of streptococci of serological groups R and S and serological groups U, V and P are found in the fourth cluster. The fifth cluster comprises strains of Streptococcus anginosus S. intermedius, Streptococcus MG and serological groups G ('minute' colony, type I) and F (type I). Most of the test strains contain the peptidoglycan type Lys-Ala_1–3. Only streptococci of serogroups R and S reveal a directly cross-linked peptidoglycan. Rhamnose was found as characteristic component of all cell wall polysaccharides. The impact of our results on the systematics of classical pyogenic streptococci will be discussed.     

THE ASSESSMENT OF THE BACTERIOLOGICAL CONDITION OF MILK BOTTLES

SUMMARY: A study of the relative values of a number of bacteriological tests for assessing the condition of milk bottles indicated that the colony count of the bottle rinse solution on yeastrel milk agar incubated for 4 days at 30°, combined with a clot-on-boiling test applied to 1 ml. of rinse in 9 ml. of sterile milk after incubation for 72 hr. at 19–20°, gave the most useful results.
The mean of the ratios of colony counts at 30° to those at 37° was 15·1, while it was as high as 22·9 for rinses with 37° of over 600 for an unsatisfactory bottle should be retained when the test is done at 30°. The thermoduric colony count of rinses of milk bottles, even when laboratory pasteurized in milk, did not provide any additional information to that given by the colony count at 30° made without pasteurization. A high proportion of the organisms in bottle rinses survived laboratory pasteurization in milk, the survival rate being highest in efficiently treated bottles.
The clot-on-boiling test gave results in general agreement with colony counts and served to indicate the potential influence of badly contaminated bottles on the keeping quality of milk placed in them. A substantial proportion of rinses with satisfactory colony counts reduced methylene blue within 48 hr. at 19–20°.
Colony counts at 37° were on the average much lower for bottles treated with steam than for bottles submitted to detergent treatment in various types of bottle washing machines. Treatment of bottles by steam or hypochlorite was more efficiently done on the farms than at the dairies.     

The effect of high temperatures on seed germination of one native and two introduced conifers in Patagonia

We examined the effect of thermal shock on the germination of seeds of three conifers, two introduced (Pseudotsuga menziesii and Pinus ponderosa), and one native to Patagonia (Araucaria araucana). Previous research has suggested increased susceptibility to invasions in burnt areas, and therefore, the effect of simulated fire (heat) on seed germination in these native and introduced species was compared. Seeds were heated to two different heat intensities (50°C and 100°C) for 1 or 5 min, which is within the temperature range reached in the upper soil layers during forest fires. Germination tests were then carried out in a growth chamber. The heat treatments had a negative effect on the germination of P. menziesii at temperatures of 100°C, and a negative effect on the germination of P. ponderosa at the temperature of 100°C and the exposure of 5 min. The heat treatments had no affect at all on A. araucana. The species with larger seeds (A. araucana) had higher survival rates after the thermal shocks. Also intraspecific differences in seed sizes possibly point at larger seeds surviving thermal shocks better than smaller seeds. In addition, thermal shock caused a delay in the onset of germination in the two introduced species, while it did not change the time for germination in A. araucana.     

The effect of combined heat and ultrasound on multicellular tumour spheroids

The effect of combined ultrasound and heat treatments on Chinese hamster multicellular spheroids of varying size was investigated using growth rate, single cell survival and ultrastructural damage as endpoints. Ultrasonic irradiation at 37 degrees C had no effect on the growth rate of 200-730 microns spheroids. Similarly there was no effect on the growth rate of 350 microns spheroids when irradiated during a 60 min exposure to 41.5 degrees C. However, spheroids of 200-700 mm diameter showed growth delay when held at 43 degrees C for 1 h. The effect was enhanced with concomitant ultrasound irradiation but was not dependent on spheroid size. When 200 and 400 microns spheroids held at 43 degrees C for 60 min were irradiated with different ultrasonic intensities a dose-dependent decrease in surviving fraction and a dose-dependent increase in growth delay was obtained. When surviving fraction was plotted as a function of growth delay a good correlation was obtained, suggesting that the combination of heat and ultrasound irradiation does not produce cytostasis in the surviving cells of either 200 or 400 microns spheroids. At the ultrastructural level increased cytoplasmic vacuolation was the only result of ultrasonic irradiation at 37 degrees C. Exposure to 43 degrees C for 60 min was required to elicit thermal damage. This took the form of membrane evagination at the spheroid surface, vacuolation of the cytoplasm, grouping of organelles around the periphery of the nucleus, and fragmentation of the nucleolus. These effects were enhanced with concomitant ultrasonic irradiation but other features were also noted, viz. disaggregation of polyribosomes, dilation of the rough endoplasmic reticulum and blebbing of the nuclear membrane. Damage was independent of spheroid size. These results are in agreement with previous data obtained from single-cell studies. Indicating that there is a non-thermal, non-cavitational component to the cell killing in multicellular spheroids resulting from combined heat and ultrasound treatment.     

The response of mouse skin to combined hyperthermia and X-rays.

The effects of combined hyperthermia and X-irradiation were studied in the skin of the mouse ear. Ears were heated for 1 hour by immersion in a waterbath at temperatures ranging from 37 degrees C--43 degrees C. These heat treatments had little visible effect alone, but when combined with X-rays, enhanced the radiation response. Enhancement depended on the degree of heating. When heat was given immediately after X-rays, the radiation dose to cause a given skin reaction had to be reduced by about 10 per cent for 37 degrees C and about 40 per cent of 43 degrees C. The timing and sequence of the two treatments were important. Heat after X-rays was less effective than heat before X-rays. When heat followed X-rays, the enhancing effect was lost completely if the interval exceeded 4 hours. When heat preceded X-rays, the effect was lost more slowly, depending on temperature. The implications of this for the treatment of cancer by combined therapy are discussed.     

Comparative study on the induction of triploidy in tilapias, using cold- and heat-shock techniques

Optimal conditions for the induction of triploidy in two different species of tilapia, Oreochromis aureus and Oreachromis niloticus , were determined using either heat-shock or cold-shock techniques. These treatments were applied at conditions that were separately determined for each species. Embryos of these two species were found to differ in their response to heat-shock, but not to cold-shock treatments. The yield of triploid O. niloticus embryos induced at 40.5°C at the zygotic age of 3-5 min did not exceed 13%, in contrast to 60% obtained in O. aureus (heat-shocked at 39.5°C at zygotic age of 3 min). The possibility that the different responses of the two species to heat shock is maternally inherited was supported by the fact that the two-way F₁ hybrids also responded in a similar maternally dominant manner. Although the induction of triploidy using heat shock was possible only within a narrow range of zygotic ages (2.5–3.5 and 3.5–4.5 min for O. aureus and O. niloticus , respectively), the induction of triploidy using cold-shock treatment (11°C for 60 min) could be achieved within a wider range of zygotic ages (0–15 min). A satisfactory yield of troploids in the two species (50 and 60% in O. niloticus and O. aureus , respectively) was obtained using this technique. The possible interpretation of these results, that triploidy could be induced by interfering at two distinct meiotic cell-division stages, is discussed. It is concluded that the cold-shock technique is more advantageous for inducing triploidy in O. niloticus , whereas both cold-shock and heat-shock techniques are equally efficient in O. aureus.     

Influence of Temperature and Pressure on the Lethality of Ultrasound

A specially designed resistometer was constructed, and the lethal effect on Yersinia enterocolitica of ultrasonic waves (UW) at different static pressures (manosonication [MS]) and of combined heat-UW under pressure treatments (manothermosonication [MTS]) was investigated. During MS treatments at 30°C and 200 kPa, the increase in the amplitude of UW of 20 kHz from 21 to 150 μm exponentially decreased decimal reduction time values (D_MS) from 4 to 0.37 min. When pressure was increased from 0 to 600 kPa at a constant amplitude (150 μm) and temperature (30°C), D_MS values decreased from 1.52 to 0.20 min. The magnitude of this decrease in D_MS declined progressively as pressure was increased. The influence of pressure on D_MS values was greater with increased amplitude of UW. Pressure alone of as much as 600 kPa did not influence the heat resistance of Y. enterocolitica (D₆₀ = 0.094; z = 5.65). At temperatures of as much as 58°C, the lethality of UW under pressure was greater than that of heat treatment alone at the same temperature. At higher temperatures, this difference disappeared. Heat and UW under pressure seemed to act independently. The lethality of MTS treatments appeared to result from the added effects of UW under pressure and the lethal effect of heat. The individual contributions of heat and of UW under pressure to the total lethal effect of MTS depended on temperature. The inactivating effect of UW was not due to titanium particles eroded from the sonication horn. The addition to the MS media of cysteamine did not increase the resistance of Y. enterocolitica to MS treatment. MS treatment caused cell disruption.     

In-vitro bacteriocin-mediated antagonism by oral streptococci against human carrier strains of staphylococci

All strains of oral streptococci tested and specially those of Streptococcus mutans, Strep. sanguis and Strep. minor produced more than one distinct bacteriocin-like substance with variable inhibitory activity on 20 indicator staphylococci. Inhibitory activity was comparatively higher on nasal strains of Staph. aureus and Staph. epidermidis than on strains of both species isolated from the mouth. Nineteen of 20 staphylococcal indicators were inhibited by 1–12 of the 12 effector streptococci. Sensitivity of nasal staphylococci to bacteriocins (frequency of positive inhibitory tests and total inhibition zone diameters) was significantly higher ( P < 0·001, χ² test and P < 0·05, t test respectively) than that of oral ones. The sensitivity of nasal over oral Staph. aureus ( P < 0·001 and P < 0·01) and of oral Staph. epidermidis over oral Staph. aureus ( P < 0·01 and P < 0·05) was also significantly higher. The evaluation of variability of inhibitory patterns of bacteriocins produced by streptococci (p-typing), of sensitivity patterns of staphylococci to bacteriocins (s-typing) and of the significantly higher sensitivity of nasal over oral staphylococci to bacteriocins from the epidemiological and ecological veiwpoints are discussed.     

Effervescent tablets and ultrasonic devices against Candida and mutans streptococci in denture biofilm

doi: 10.1111/j.1741‐2358.2010.00378.x Effervescent tablets and ultrasonic devices against Candida and mutans streptococci in denture biofilm Objective: To evaluate the antimicrobial action of effervescent tablets and ultrasound on Candida spp. and mutans streptococci from denture biofilm. Background: It is not uncommon for edentulous patients to be elderly and find it difficult to brush their dentures. Hence, auxiliary methods are required for cleansing dentures as well as treating oral infections. Materials and methods: Seventy‐seven complete denture wearers were randomly assigned into four groups: (A) Brushing with water (control); (B) Effervescent tablets; (C) Ultrasonic device (Ultrasonic Cleaner, model 2840 D); (D) Effervescent tablets and ultrasonic device. All groups brushed their dentures with a specific brush and water, three times a day, before applying their treatments. Denture biofilm was collected at baseline and after 21 days. The samples were collected by brushing the dentures with saline and the detached microbial cells were quantified by plating. Counts [log (CFU+1) ml^?1] of total aerobes, Candida spp. and mutans streptococci were compared by one‐way anova or Kruskal–Wallis test (α = 0.05). Results: No significant difference was found among the methods from C. albicans (p = 0.76), C. tropicalis (p = 0.94) and C. glabrata (p = 0.80). Lower counts were found for methods B and D when compared with the other methods against mutans streptococci (p < 0.001). Method B showed lower total aerobic counts than A, whereas C and D showed intermediate results (p = 0.011). Conclusion: The effervescent tablets significantly reduced mutans streptococci and total aerobes from denture biofilm. However, they was not as effective against C. albicans. Ultrasonic cleansing presented a discrete antimicrobial effect and was less effective than the tablets for complete denture disinfection.     

Septage treatments to reduce the numbers of bacteria and polioviruses

Disposal of the pumped contents of septic tanks (septage) represents a possible means of dissemination of enteric pathogens including viruses, since persistence of enteroviruses in septic tank sludge for greater than 100 days has been demonstrated. The risk of exposure to potentially infectious agents can be reduced by disinfecting septages before their disposal. Of the septage disinfectants examined (technical and analytical grade glutaraldehyde, hydrogen peroxide, heat treatments, and a combination of heat and hydrogen peroxide), the treatment including hydrogen peroxide (5 mg, plus 0.33 mg of trichloroacetic acid, per ml of septage) and 55 degrees C killed virtually all the bacteria in septage within 1 h, whereas 55 degrees C alone inactivated inoculated polioviruses within 30 min. Virus was the most sensitive to heat, whereas fecal coliforms appeared to be the most sensitive to all chemical treatments. The responses of fecal streptococci and virus to both grades of glutaraldehyde (each at 1 mg/ml) were similar. Virus was more resistant than either fecal streptococci or total bacteria to low concentrations of hydrogen peroxide (1 to 5 mg/ml); however, virus and fecal streptococci were more labile than total bacteria to the highest peroxide concentration (10 mg/ml) examined. It is possible that the treatment combining heat and hydrogen peroxide was the most effective in reducing the concentrations of all bacteria, because catalase and peroxidases as well as other enzymes were heat inactivated, although catalase seems the most likely cause of damage. However, this most effective treatment does not appear to be practical for on-site use as performed, so further work on septage disinfection is recommended.     

Septage treatments to reduce the numbers of bacteria and polioviruses.

Disposal of the pumped contents of septic tanks (septage) represents a possible means of dissemination of enteric pathogens including viruses, since persistence of enteroviruses in septic tank sludge for greater than 100 days has been demonstrated. The risk of exposure to potentially infectious agents can be reduced by disinfecting septages before their disposal. Of the septage disinfectants examined (technical and analytical grade glutaraldehyde, hydrogen peroxide, heat treatments, and a combination of heat and hydrogen peroxide), the treatment including hydrogen peroxide (5 mg, plus 0.33 mg of trichloroacetic acid, per ml of septage) and 55 degrees C killed virtually all the bacteria in septage within 1 h, whereas 55 degrees C alone inactivated inoculated polioviruses within 30 min. Virus was the most sensitive to heat, whereas fecal coliforms appeared to be the most sensitive to all chemical treatments. The responses of fecal streptococci and virus to both grades of glutaraldehyde (each at 1 mg/ml) were similar. Virus was more resistant than either fecal streptococci or total bacteria to low concentrations of hydrogen peroxide (1 to 5 mg/ml); however, virus and fecal streptococci were more labile than total bacteria to the highest peroxide concentration (10 mg/ml) examined. It is possible that the treatment combining heat and hydrogen peroxide was the most effective in reducing the concentrations of all bacteria, because catalase and peroxidases as well as other enzymes were heat inactivated, although catalase seems the most likely cause of damage. However, this most effective treatment does not appear to be practical for on-site use as performed, so further work on septage disinfection is recommended.