Survival Times of Selected Enteropathogenic Bacteria in Frozen Passionfruit Nectar Base

Five test organisms were used: Escherichia coli, Salmonella typhosa, Salmonella schottmuelleri, Salmonella enteritidis, and Shigella paradysenteriae. Even when large inocula of these test cultures were introduced into fresh passionfruit nectar base, all test organisms were killed within 1 to 2 hr, provided the nectar base was held at room temperature for more than 1 hr before freezing. If the nectar base was frozen immediately after inoculation, four of the five test organisms were eliminated almost as quickly. But the fifth, Salmonella enteritidis, proved to be exceptional: it was being recovered after 90 days of storage at -20 C, when the last available sample was analyzed.     

Deoxyribonucleic Acid Base Composition and Biochemical Properties of Certain Coagulase-Negative Enterotoxigenic Cocci

Eight coagulase-negative, enterotoxigenic strains of cocci and one weakly coagulase-positive strain isolated from a number of different sources, including cases of food poisoning incidents, were evaluated for their relationship to Staphylococcus aureus on the basis of deoxyribonucleic acid (DNA) buoyant density and physiological studies. One strain of cocci produced enterotoxins A and C, two strains produced types B and C, four strains produced only type C, and one strain only type D. The enterotoxin produced by one strain of cocci was serologically untypable. None of the test organisms produced detectable amounts of enterotoxin in broth cultures. The test strains of cocci exhibited the following profile: all produced catalase; all grew anaerobically and fermented glucose; five were sensitive to lysostaphin; the percentage of guanine plus cytosine content of their DNA varied from 32.7 to 37.6; five produced acid from mannitol both aerobically and anaerobically; two formed δ-hemolysin; five produced phosphatase and acetoin; and all produced heat-stable nuclease. None of the organisms exhibited typical characteristics of S. aureus, S. epidermidis, or S. saprophyticus. On the basis of the present data and data reported elsewhere, these organisms should be considered as variants or mutants of S. aureus.     

Reversible thermal unfolding of thermostable cytochrome c-552.

Reversible thermal denaturation of cytochrome c-552 from the extremely thermophilic bacterium Thermus thermophilus was studied by circular dichroism and fluorescence spectroscopy. Thermal denaturation in the presence of guanidine hydrochloride is completely reversible. The thermodynamic parameters for the reaction have been calculated based on a two-state mechanism. The free energy change on denaturation (ΔG) at 25 °C in the absence of denaturant is estimated to be 28.5 ± 0.15 kcal/mol, which is larger than that of cytochrome c from mesophilic organisms. The temperature of maximum stability is approximately 27 °C, which is higher than those of cytochromes c from mesophilic organisms (9 to 12 °C). The temperature dependences of the enthalpy and entropy changes are similar to those of cytochromes c from mesophilic organisms. The heat capacity change on denaturation is between 1250 and 1680 cal/deg mole, which is similar to those of cytochromes c from mesophilic organisms (1500 to 2500 cal/deg mol). From these results, it has been concluded that T. thermophilus cytochrome c is more stable than cytochromes c from mesophilic organisms by virtue of the fact that the free energy change for denaturation is greater and has its maximum at a higher temperature.     

Scanning Electron Microscopy of Colonies of Six Species of Candida

Thirty strains of six species of Candida isolated from patients were cultured for 60 h on Sabouraud agar, freeze-dried, and examined with a scanning electron microscope. The colonies were circular (Candida albicans, C. guilliermondii) or oval (C. tropicalis, C. pseudotropicalis, C. krusei, C. parakrusei) in outline, and those of C. pseudotropicalis and C. krusei had an irregular outline due to a peripheral pseudomycelium. The morphology of individual microorganisms was examined at the margins and apex of those species which lacked a surface coat (C. pseudotropicalis, C. krusei, C. parakrusei, C. guilliermondii), and through cracks in the surface coating of those which showed a surface coat (C. albicans, C. tropicalis). All species showed buds, bud scars, and interconnecting intercellular processes, but were generally spherical (C. albicans, C. tropicalis) or ovoid (C. pseudotropicalis, C. krusei, C. parakrusei, C. guilliermondii) in fixed preparations. In unfixed material, individual organisms were almost invariably indented. Fixation with 3% glutaraldehyde and washing before freeze-drying caused partial removal of the surface coating of colonies of C. albicans and C. tropicalis, which persisted only as irregular sheets or as a filamentous meshwork. This filamentous meshwork was also present among the organisms of colonies of C. albicans, C. tropicalis, and C. pseudotropicalis. It is concluded that these filaments represent the precipitation or unmasking of some component of the intercellular matrix of these organisms.     

Inhibition of microorganisms by topical anesthetics

The effect of various topical anesthetics and their preservatives on the growth of Pseudomonas aeruginosa, Staphyloccoccus albus, and Candida albicans was investigated. The topical anesthetics were proparacaine HCl, tetracaine HCl, cocaine HCl, and benoxinate HCl. The preservatives were chlorobutanol and butyl p-hydroxybenzoate. Proparacaine inhibited C. albicans but not P. aeruginosa or S. albus. All three test organisms were inhibited to varying degrees by tetracaine, benoxinate, cocaine, chlorobutanol, and butyl p-hydroxybenzoate.     

Comparative total and proportional rate of germination of Bipolaris sorokiniana and Curvularia geniculata conidia is influenced by culture age and temperature

Comparative observations of Bipolaris sorokiniana and Curvularia geniculata conidia germination as influenced by culture age and temperature showed some distinct differences, but generally established the ability of these organisms to function under similar conditions. Total germination of B. sorokiniana conidia was favored by increasing culture age from 20 to 60 days and temperature to 25°C; total conidia germination of C. geniculata was favored by increasing temperature to 25°C, but increasing culture age decreased germination. These reactions seem associated with conidia age. Maximum proportional intra-population germination of conidia of each organism also varied with culture age and temperature. At temperatures of 5°C and 15°C, amplitude of maximum proportional germination of both organisms increased as culture age was increased from 20 to 40 days and then decreased among 60-day-old cultures. At 25°C and above, amplitude of maximum proportional germination of conidia of both organisms decreased from each older culture. Progressively increasing temperature at a given culture age increased the amplitude of maximum proportional germination up to 25°C for conidia of B. sorokiniana, but generally decreased it for conidia of C. geniculata (except 20-day-old cultures). Frequency (specific 2 h interval) of maximum proportional intrapopulation germination of B. sorokiniana shifted from 6 h to 2 h in response to increasing temperature and culture age; conidia from youngest cultures of C. geniculata shifted to intervals of 4 h and 2 h in response to increasing temperature to 25°C, but among conidia from 60-day-old cultures, frequency shifted to 6 h intervals at all temperatures. Above 25°C, maximum proportional germination of C. geniculata conidia from cultures of all ages occurred at 6 h. It was concluded that the germination response of B. sorokiniana and C. geniculata conidia to temperature and culture age (and, subsequently, conidia age) are enough similar that these organisms could function in a potential ‘disease complex’ on Poa pratensis and Agrostis palustris.     

Effect of Chlorine Dioxide Gas on Fungi and Mycotoxins Associated with Sick Building Syndrome

The growth of indoor molds and their resulting products (e.g., spores and mycotoxins) can present health hazards for human beings. The efficacy of chlorine dioxide gas as a fumigation treatment for inactivating sick building syndrome-related fungi and their mycotoxins was evaluated. Filter papers (15 per organism) featuring growth of Stachybotrys chartarum, Chaetomium globosum, Penicillium chrysogenum, and Cladosporium cladosporioides were placed in gas chambers containing chlorine dioxide gas at either 500 or 1,000 ppm for 24 h. C. globosum was exposed to the gas both as colonies and as ascospores without asci and perithecia. After treatment, all organisms were tested for colony growth using an agar plating technique. Colonies of S. chartarum were also tested for toxicity using a yeast toxicity assay with a high specificity for trichothecene mycotoxins. Results showed that chlorine dioxide gas at both concentrations completely inactivated all organisms except for C. globosum colonies which were inactivated an average of 89%. More than 99% of ascospores of C. globosum were nonculturable. For all ascospore counts, mean test readings were lower than the controls (P < 0.001), indicating that some ascospores may also have been destroyed. Colonies of S. chartarum were still toxic after treatment. These data show that chlorine dioxide gas can be effective to a degree as a fumigant for the inactivation of certain fungal colonies, that the perithecia of C. globosum can play a slightly protective role for the ascospores and that S. chartarum, while affected by the fumigation treatment, still remains toxic.     

Emerging findings from studies of phospholipase D in model organisms (and a short update on phosphatidic acid effectors)

Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to generate phosphatidic acid and choline. Historically, much PLD work has been conducted in mammalian settings although genes encoding enzymes of this family have been identified in all eukaryotic organisms. Recently, important insights on PLD function are emerging from work in yeast, but much less is known about PLD in other organisms. In this review we will summarize what is known about phospholipase D in several model organisms, including C. elegans, D. discoideum, D. rerio and D. melanogaster. In the cases where knockouts are available (C. elegans, Dictyostelium and Drosophila) the PLD gene(s) appear not to be essential for viability, but several studies are beginning to identify pathways where this activity has a role. Given that the proteins in model organisms are very similar to their mammalian counterparts, we expect that future studies in model organisms will complement and extend ongoing work in mammalian settings. At the end of this review we will also provide a short update on phosphatidic acid targets, a topic last reviewed in 2006.     

Toxicity of zinc to schistosoma Mansoni Cercariae in a Chemically Defined Water Medium

Time-until-death studies were run on cercariae of Schistosoma mansoni in 15 concentrations of zinc (from zinc sulfate) ranging from 57.6 ppm to 1 ppb. A chemically defined water medium was used for the dilution and control medium, and a partially enclosed slide chamber was perfected for the detailed observation of test cercariae. In concentrations of less than 57.6 ppm, zinc was found to have little lethal effect on cercariae during that period in which these larvae were most likely to be infective (0–6 hours) after emergence. Zinc concentration of 1 ppb killed all test cercariae within 46 hours at 21–22°C.At the highest concentration tested, 57.6 ppm zinc immobilized all test organisms within 6 hours at 21–2°C. The average time for 100 percent mortality for the control was 49 hours. The cercaricidal effects of zinc were shown to be insignificant in concentrations that would not be detrimental to other aquatic organisms.     

Transformation of Sexually Transmitted Infection-Causing Serovars of Chlamydia trachomatis Using Blasticidin for Selection

Plasmid-free Chlamydia trachomatis serovar L2 organisms have been transformed with chlamydial plasmid-based shuttle vectors pGFP::SW2 and pBRCT using β-lactamase as a selectable marker. However, the recommendation of amoxicillin, a β-lactam antibiotics, as one of the choices for treating pregnant women with cervicitis due to C. trachomatis infection has made the existing shuttle vectors unsuitable for transforming sexually transmitted infection (STI)-causing serovars of C. trachomatis. Thus, in the current study, we modified the pGFP::SW2 plasmid by fusing a blasticidin S deaminase gene to the GFP gene to establish blasticidin resistance as a selectable marker and replacing the β-lactamase gene with the Sh ble gene to eliminate the penicillin resistance. The new vector termed pGFPBSD/Z::SW2 was used for transforming plasmid-free C. trachomatis serovar D organisms. Using blasticidin for selection, stable transformants were obtained. The GFP-BSD fusion protein was detected in cultures infected with the pGFPBSD/Z::SW2-trasnformed serovar D organisms. The transformation restored the plasmid property to the plasmid-free serovar D organisms. Thus, we have successfully modified the pGFP::SW2 transformation system for studying the biology and pathogenesis of other STI-causing serovars of C. trachomatis.     

Usefulness of Electrophoretic Pattern of Cell Envelope Protein as a Taxonomic Tool for Fishhold Slime Moraxella Species

Nine independent Moraxella cultures were isolated from the accumulated slime in fishholds of fishery trawlers. It is significant that none of these isolates was viable above 30°C, a temperature well below the usual incubation temperature for plate counts of food samples. The traditional taxonomic parameters showed no significant dissimilarities among these closely related marine organisms or between them and conventional moraxellas. However, cell envelope protein profiles examined on sodium dodecyl sulfate-polyacrylamide gels revealed that the organisms fell into several distinct groups. The cell envelope protein profile could be a simple and quick test to determine the fine relationships between individual isolates.     

Metabolic Commensalism and Competition in a Two-Species Microbial Consortium

We analyzed metabolic interactions and the importance of specific structural relationships in a benzyl alcohol-degrading microbial consortium comprising two species, Pseudomonas putida strain R1 and Acinetobacter strain C6, both of which are able to utilize benzyl alcohol as their sole carbon and energy source. The organisms were grown either as surface-attached organisms (biofilms) in flow chambers or as suspended cultures in chemostats. The numbers of CFU of P. putida R1 and Acinetobacter strain C6 were determined in chemostats and from the effluents of the flow chambers. When the two species were grown together in chemostats with limiting concentrations of benzyl alcohol, Acinetobacter strain C6 outnumbered P. putida R1 (500:1), whereas under similar growth conditions in biofilms, P. putida R1 was present in higher numbers than Acinetobacter strain C6 (5:1). In order to explain this difference, investigations of microbial activities and structural relationships were carried out in the biofilms. Insertion into P. putida R1 of a fusion between the growth rate-regulated rRNA promoter rrnBP1 and a gfp gene encoding an unstable variant of the green fluorescent protein made it possible to monitor the physiological activity of P. putida R1 cells at different positions in the biofilms. Combining this with fluorescent in situ hybridization and scanning confocal laser microscopy showed that the two organisms compete or display commensal interactions depending on their relative physical positioning in the biofilm. In the initial phase of biofilm development, the growth activity of P. putida R1 was shown to be higher near microcolonies of Acinetobacter strain C6. High-pressure liquid chromatography analysis showed that in the effluent of the Acinetobacter strain C6 monoculture biofilm the metabolic intermediate benzoate accumulated, whereas in the biculture biofilms this was not the case, suggesting that in these biofilms the excess benzoate produced by Acinetobacter strain C6 leaks into the surrounding environment, from where it is metabolized by P. putida R1. After a few days, Acinetobacter strain C6 colonies were overgrown by P. putida R1 cells and new structures developed, in which microcolonies of Acinetobacter strain C6 cells were established in the upper layer of the biofilm. In this way the two organisms developed structural relationships allowing Acinetobacter strain C6 to be close to the bulk liquid with high concentrations of benzyl alcohol and allowing P. putida R1 to benefit from the benzoate leaking from Acinetobacter strain C6. We conclude that in chemostats, where the organisms cannot establish in fixed positions, the two strains will compete for the primary carbon source, benzyl alcohol, which apparently gives Acinetobacter strain C6 a growth advantage, probably because it converts benzyl alcohol to benzoate with a higher yield per time unit than P. putida R1. In biofilms, however, the organisms establish structured, surface-attached consortia, in which heterogeneous ecological niches develop, and under these conditions competition for the primary carbon source is not the only determinant of biomass and population structure.     

Combined Effects of Ultrahigh Vacuum and Temperature on the Viability of Some Spores and Soil Organisms

Considerably fewer spores of Bacillus stearothermophilus, B. megaterium, and Clostridium sporogenes were recovered than were spores of B. subtilis var. niger and Aspergillus niger after 4 to 5 days at 53 and 60 C in ultrahigh vacuum. There were no significant differences in the recoveries of these five organisms at 25 C and atmospheric pressure, and after exposure to 25 and -190 C in vacuum. At 60 C, a far greater decrease in viability was demonstrated for B. stearothermophilus, B. megaterium, and C. sporogenes in ultrahigh vacuum than at atmospheric pressure. Viable B. subtilis var. niger spores were not detected in an initial 10⁷ spores after retention at 90 C and ultrahigh vacuum, and 10⁴ spores were viable after 5 days at 90 C and atmospheric pressure from an initial 10⁶ spores. Molds and actinomycetes in soil were particularly resistant up to 69 C in vacuum. Actinomycetes were the only soil organisms recovered so far at 120 C.     

Effects of storage temperature,light and time on stability of triple sugar iron agar and its productivity forEscherichia coli andSalmonella typhimurium

Laboratory evaluation showed a similarity between prepared ready for-use triple sugar iron agar (TSI) medium and the Oxoid form with reference to the enumeration of five test organisms related to the familyEnterobactenaceae. Storage of the prepared ready-for-use TSI medium at 20–22 °C should not exceed 6 months Lowering the storage temperature to 10 °C raised the period of storage to two years. Combination of darkness and low temperature (10 to 20 °C) represents the best conditions for storage of the TSI medium in laboratories. Dark colour ampoules can extend the expiry date of the product     

Oviduct Infection and Hydrosalpinx in DBA1/j Mice Is Induced by Intracervical but Not Intravaginal Inoculation with Chlamydia muridarum

Intravaginal infection with C. muridarum in mice often results in hydrosalpinx similar to that found in women urogenitally infected with C. trachomatis, making the C. muridarum lower genital tract infection murine model suitable for studying C. trachomatis pathogenesis. To our surprise, DBA1/j mice were highly resistant to hydrosalpinx following an intravaginal infection with C. muridarum although these mice were as susceptible to lower genital tract infection as other mouse strains. A significantly lower level of C. muridarum organisms was recovered from the oviduct of DBA1/j mice, correlating the resistance to hydrosalpinx with reduced ascension of C. muridarum to the oviduct. The DBA1/j resistance to hydrosalpinx was effectively overcome by intracervical inoculation with C. muridarum. The intracervically inoculated DBA1/j mice developed severe hydrosalpinx with the highest levels of live C. muridarum organisms recovered from uterine tissue on day 3 and oviduct tissue on day 7 post inoculation while in intravaginally inoculated DBA1/j mice, the peak of live organism recovery from uterine tissue was delayed to day 7 with no rise in the amount of live organisms recovered from the oviduct. These observations have not only validated the correlation between hydrosalpinx and live organism invasion in the oviduct but also demonstrated that the intracervical inoculation, by promoting rapid chlamydial replication in the uterine epithelial cells and ascension to the oviduct of DBA1/j mice, may be used for further understanding chlamydial pathogenic mechanisms. The above findings also suggest that strategies aimed at reducing tubal infection may be most effective in blocking tubal pathology.     

Compound-Specific Isotopic Fractionation Patterns Suggest Different Carbon Metabolisms among Chloroflexus-Like Bacteria in Hot-Spring Microbial Mats

Stable carbon isotope fractionations between dissolved inorganic carbon and lipid biomarkers suggest photoautotrophy by Chloroflexus-like organisms in sulfidic and nonsulfidic Yellowstone hot springs. Where co-occurring, cyanobacteria appear to cross-feed Chloroflexus-like organisms supporting photoheterotrophy as well, although the relatively small ¹³C fractionation associated with cyanobacterial sugar biosynthesis may sometimes obscure this process.     

Manipulation of host behaviour by parasites: ecosystem engineering in the intertidal zone?

Understanding the influence of parasites on the community ecology of free-living organisms is an emerging theme in ecology. The cockle Austrovenus stutchburyi is an abundant mollusc inhabiting the sheltered shores of New Zealand. This species, which lives just few centimetres under the surface, plays a key role for many benthic invertebrate species, because in these habitats the cockle shell is the only available hard surface where invertebrates can establish. However, the behaviour of this cockle can be altered locally by a parasite, the trematode Curtuteria australis. Indeed, heavily infected cockles are unable to bury perfectly and typically lie entirely exposed at the surface of the mud. In this study, we investigated the ecological consequences of this behavioural alteration for two invertebrates species commonly associated with cockles, the anemone Anthopleura aureoradiata and the limpet Notoacmea helmsi. A field study first demonstrated that in both infected and non-infected populations of cockles, there was a negative relationship between the number of anemones and limpets found on cockles. In the laboratory, we showed that predation of limpets by anemones is possible when they share the same cockle shell. In a heavily infected population of cockles, limpets were significantly more frequent and more abundant on cockles manipulated by C. australis than on cockles with a normal behaviour. A colonization test conducted in natural conditions demonstrated that the predominance of limpets on manipulated cockles results from a direct habitat preference. Conversely, anemones were significantly less frequent and less abundant on manipulated cockles than on cockles manipulated by C. australis. A desiccation test revealed that, relative to limpets, they had a lower resistance to this physical stress. We discuss our results in relation to current ideas on ecosystem engineering by organisms.     

Thermal Resistance of Salmonellae Isolated from Dry Milk

Salmonella anatum, S. binza, S. cubana, S. meleagridis, S. newbrunswick, and S. tennessee isolated from dry milk, and S. senftenberg 775W were studied for heat resistance to determine whether these organisms would survive pasteurization as recommended by the 1965 Pasteurized Milk Ordinance of the U.S. Public Health Service. Thermal inactivation determinations were made on washed cells of the test microorganisms suspended in sterile whole milk. Excluding S. senftenberg, D values ranged from 3.6 to 5.7 sec at 62.8 C, from 1.1 to 1.8 sec at 65.6 C, and from 0.28 to 0.52 sec at 68.3 C. Corresponding values for S. senftenberg were 34.0, 10.0, 1.2, and 0.55 sec for respective exposure temperatures of 65.5, 68.3, 71.7, and 73.9 C. The present milk pasteurization processes as recommended by the Public Health Service will inactivate all seven strains of salmonellae studied, provided that the initial concentration does not exceed a calculated 3 × 10¹² salmonellae per ml of milk.     

Verification of immunosuppression in chicks caused by Cryptosporidium baileyi infection using Brucella abortus strain 1119-3

Humoral immune response of young chicks to Brucella abortus strain 1119-3 inoculation was monitored to verify the degree of immunosuppression caused by infection with Cryptosporidium baileyi. Young chicks (2-day-old) were orally inoculated each with 2 × 10⁶ oocysts of C. baileyi, and then injected intramuscularly with 0.3 ml B. abortus strain 1119-3 containing 1 × 10⁹ living organisms on day 14 postinoculation (PI). Serum samples were tested by plate agglutination test on day 17 PI onwards at an interval of 3-6 days over a period of 36 days. Infected chicks with the coccidium showed significantly lower antibody titers than those of uninfected controls (P < 0.05). These findings document that C. baileyi infection in early life stage may predispose chicks easily to other potential poultry diseases.