Performance of Media for Recovering Stressed Cells of Enterobacter sakazakii as Determined Using Spiral Plating and Ecometric Techniques

A study was done to determine the performance of differential, selective media for supporting resuscitation and colony development by stressed cells of Enterobacter sakazakii. Cells of four strains of E. sakazakii isolated from powdered infant formula were exposed to five stress conditions: heat (55°C for 5 min), freezing (−20°C for 24 h, thawed, frozen again at −20°C for 2 h, thawed), acidic pH (3.54), alkaline pH (11.25), and desiccation in powdered infant formula (water activity, 0.25; 21°C for 31 days). Control and stressed cells were spiral plated on tryptic soy agar supplemented with 0.1% pyruvate (TSAP, a nonselective control medium); Leuschner, Baird, Donald, and Cox (LBDC) agar (a differential, nonselective medium); Oh and Kang agar (OK); fecal coliform agar (FCA); Druggan-Forsythe-Iversen (DFI) medium; violet red bile glucose (VRBG) agar; and Enterobacteriaceae enrichment (EE) agar. With the exception of desiccation-stressed cells, suspensions of stressed cells were also plated on these media and on R&F Enterobacter sakazakii chromogenic plating (RF) medium using the ecometric technique. The order of performance of media for recovering control and heat-, freeze-, acid-, and alkaline-stressed cells by spiral plating was TSAP > LBDC > FCA > OK, VRBG > DFI > EE; the general order for recovering desiccated cells was TSAP, LBDC, FCA, OK > DFI, VRBG, EE. Using the ecometric technique, the general order of growth indices of stressed cells was TSAP, LBDC > FCA > RF, VRBG, OK > DFI, EE. The results indicate that differential, selective media vary greatly in their abilities to support resuscitation and colony formation by stressed cells of E. sakazakii. The orders of performance of media for recovering stressed cells were similar using spiral plating and ecometric techniques, but results from spiral plating should be considered more conclusive.     

两株耐碱酵母的pH耐受实验观察

对分离鉴定的两株酵母菌用比浊法和Bradford法进行pH耐受范围检测。Trichosporon asahiiXJU-1的pH耐受范围为2.0~13.0,最适pH值为8.0。Rhodotorula mucilaginosaXJU-1的pH耐受范围为3.0~12.0,最适pH值为8.5。比浊法在pH4.0~10.0测出数值比较可靠,而在pH<4.0和pH≥10.0产生了较大误差。在最适pH时,Bradford法间接测定两酵母的生长量,T.asahii总溶解蛋白为290μg/mL,Rh.mucilaginosa总溶解蛋白为164μg/mL。Bradford法在pH2.0~13.0范围均能较准确地反映出菌株生长状况,数据表明两株酵母有广阔的pH耐受性,它们是耐碱酵母菌的新成员。     

Low pH value of pericellular medium as a factor limiting cell proliferation in dense cultures

An attempt was made to determine the role of metabolic acidification of pericellular medium in regulating cell proliferation. A method of measuring the pH of pericellular medium at a distance exceeding a Debye radius of 5-10 A from the cell surface (pHp) was developed. The values of pHp and pH measured in the medium at a distance of greater than 1 cm from cells (pHm) were found to differ, depending on the cell population density. At a density of at least 7 X 10(5) cells/cm2 (maximum saturation density) and at pHm 7.4-7.6, pHp reached a value of approximately 6.5. It was found that pHm 6.5 was unfavorable for cell proliferation in sparse cultures, where pHm and pHp were equal. Based on these findings, low pHp as revealed in the present work using dense cultures at optimal pHm can be considered to be a limiting factor for cell multiplication.     

Newly Developed Sarcopenia as a Prognostic Factor for Survival in Patients who Underwent Liver Transplantation

Introduction

The relationship between a perioperative change in sarcopenic status and clinical outcome of liver transplantation (LT) is unknown. We investigated whether post-LT sarcopenia and changes in sarcopenic status were associated with the survival of patients.

Method

This retrospective study was based on a cohort of 145 patients from a single transplant center who during a mean of 1 year after LT underwent computed tomography imaging evaluation. The cross-sectional area of the psoas muscle of LT patients was compared with that of age- and sex-matched healthy individuals. The Cox proportional hazards regression model was used to determine whether post-LT sarcopenia and changes in sarcopenic status affect post-LT survival.

Results

The mean age at LT of the 116 male and 29 female patients was 50.2 ± 7.9 years; the mean follow-up duration was 51.6 ± 32.9 months. All pre-LT patients with sarcopenia still had sarcopenia 1 year after LT; 14 (15%) patients had newly developed sarcopenia. The mean survival duration was 91.8 ± 4.2 months for non-sarcopenic patients and 80.0 ± 5.2 months for sarcopenic patients (log-rank test, p = 0.069). In subgroup analysis, newly developed sarcopenia was an independent negative predictor for post-LT survival (hazard ratio: 10.53, 95% confidence interval: 1.37–80.93, p = 0.024).

Conclusion

Sarcopenia in LT recipients did not improve in any of the previously sarcopenic patients and newly developed within 1 year in others. Newly developed sarcopenia was associated with increased mortality. Newly developed sarcopenia can be used to stratify patients with regard to the risk of post-LT mortality.     

Yeasts as models in evolutionary biology

A report of the workshop 'Evolutionary and Environmental Genomics of Yeasts', Heidelberg, Germany, 1-5 October 2008.     

Pigment Epithelium-Derived Factor Is a Survival Factor for Cerebellar Granule Cells in Culture

Abstract: Pigment epithelium-derived factor (PEDF), purified from human fetal retinal pigment epithelium cell culture medium, was shown to potentiate the differentiation of human Y-79 retinoblastoma cells. To investigate potential neurotrophic effects of PEDF on neurons other than those of retinal derivation, we used cultures of cerebellar granule cells. The number of cerebellar granule cells was significantly larger in the presence of PEDF, as demonstrated by an assay for viable cells that uses 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H -tetrazolium, inner salt, conversion, by cell count, and by immunocytochemistry. The effect of PEDF showed a dose-response relationship, with a larger effect in chemically defined medium than in serum-containing medium [ED₅₀ = 30 ng/ml (0.70 n M ) in chemically defined medium and 100 ng/ml (2.3 n M ) in serum-containing medium]. PEDF had no effect on incorporation of bromodeoxyuridine (cell proliferation) or on neurofilament content (neurite outgrowth) measured by an enzyme-linked immunoadsorbent assay. These results demonstrate that PEDF has a neurotrophic survival effect on cerebellar granule cells in culture and suggest the possibility that it may affect other CNS neurons as well.     

Application of biological activated carbon as a low pH biofilter medium for gas mixture treatment

Packing material is a crucial component of a bioreactor as it is the microbial population's habitat. This study assessed potential improvements to current biofiltration processes by investigating use of a novel support medium. Biological activated carbon (BAC) with microorganisms growing on granular activated carbon can produce a novel medium in which both adsorption and biodegradation contribute to pollutants removal. Investigation of carbon characteristics demonstrated that BAC was an ideal packing medium for biofiltration. The application of the novel packing medium for gas mixture treatment was evaluated in a low pH biofilter. Results demonstrated that BAC biofilter obtained high removal efficiency for both H(2)S and toluene. The removal mechanisms of BAC were investigated after the biofilter operation and it demonstrated that the performance of the BAC system was mainly controlled by the additive contributions of two removal mechanisms - adsorption and biodegradation. This study also indicated the potential for simultaneous treatment of hydrogen sulfide and toluene at low pH condition.     

Survival of Wolbachia pipientis in cell-free medium

Wolbachia pipientis is an obligate intracellular bacterium found in a wide range of invertebrate taxa. While over ecological timescales Wolbachia infections are maintained by strict maternal inheritance, horizontal transfer events are common over evolutionary time. To be horizontally transferred between organisms, Wolbachia bacteria must pass through and survive an extracellular phase. We used BacLight live-dead staining, PCR, and fluorescence in situ hybridization to assess the ability for purified Wolbachia bacteria to survive in cell-free media. We found that purified Wolbachia bacteria were able to survive extracellularly for up to 1 week with no decrease in viability. While no replication was observed in the extracellular phase, purified Wolbachia bacteria were able to reinvade cells and establish stable infections at all time points. The ability of Wolbachia bacteria to survive outside host cells may increase the probability of successful horizontal transfer and the exploitation of new ecological niches. Our development of methods to purify and maintain viable Wolbachia bacteria from cultured cells will be useful for other researchers studying Wolbachia biology.     

Breeding Success Affects the Apparent Survival of Grassland Passerines

The apparent adult survival rate is one of the key population parameters of migratory birds. The widely used Cormack–Jolly–Seber capture–mark–recapture model has a number of disadvantages, the main one of which is the impossibility of discerning mortality and permanent emigration. The accuracy of survival estimates can be increased using a multistate capture–mark–recapture model, with the help of which it is possible to assess the survival of successful and unsuccessful birds separately. We used this model to estimate the apparent survival rates of adults in local populations of three ground-nesting passerines: Booted Warbler (Iduna caligata), Whinchat (Saxicola rubetra), and Yellow Wagtail (Motacilla flava), all breeding on abandoned agricultural lands. We studied the reproductive success of 472 marked pairs and analyzed individual capture histories of 814 birds. The previous reproductive success was found to influence significantly the apparent survival of adults. This relation was best expressed in the Yellow Wagtail (apparent survival of successful birds, φ = 0.39 ± 0.06, vs. that of unsuccessful birds, φ = 0.19 ± 0.06) and the Whinchat (apparent survival of successful birds, φ = 0.32 ± 0.05, vs. apparent survival of unsuccessful birds, φ = 0.10 ± 0.05), but a little lower in the Booted Warbler (apparent survival of successful birds, φ = 0.33 ± 0.17, vs. apparent survival of unsuccessful birds, φ = 0.16 ± 0.13). Unsuccessful individuals leave the study area for good, while most of the successful birds return there the next year. Thus, the apparent survival rate of passerines evaluated with capture–recapture models is determined to a considerable degree by the previous reproductive success within local populations.     

Estimates of Apparent Survival Rates for Forest Birds in Eastern Ecuador

Knowledge of survival rates of Neotropical landbirds remains limited, with estimates of apparent survival available from relatively few sites and species. We used capture–mark–recapture models to estimate apparent survival of 31 species from eastern Ecuador based on data collected from 2001 to 2006. Models assuming constant survival had highest support for 27 species; models incorporating effects of transients were highest for four. Average apparent survival across 30 species of passerines was 0.58 (± 0.02 SE); apparent survival was lower during the first interval after initial capture (φ₁: mean = 0.49 ± 0.03) than during subsequent intervals (φ₂: mean = 0.60 ± 0.02). Apparent survival was similar among three families represented by at least four species (Thamnophilidae: 0.57 ± 0.03, N = 10; Furnariidae: 0.59 ± 0.03, N = 5; Pipridae: 0.56 ± 0.02, N = 4). There was no indication that species that occur in flocks had higher survival than nonflocking species (obligate flock members: 0.57 ± 0.03, N = 10; facultative flock members: 0.56 ± 0.04, N = 5; nonflocking: 0.59 ± 0.03, N = 15). Comparisons of published estimates of apparent survival of tropical species demonstrated substantial differences among species and, in some cases, within species across different sites in the Neotropics. Our results support previous studies that concluded that early estimates of high (> 85%) survival in tropical birds may not be representative of all tropical species. Future studies should focus on understanding factors ( e.g. , life-history traits) that promote differences in survival among species within tropical forests.     

Effect of Relative Humidity on Survival of Candida albicans and Other Yeasts

Individual blastospores of Candida albicans were deposited on the surface of 50-mm membranes (Millipore Corp.) and placed within sealed glass chambers at various relative humidities (RH). After 48 hr, virtually all cells maintained at 100 and 10% RH had survived, but 84% of the cells maintained at 60% RH failed to develop into colonies when transferred to Sabouraud medium. No morphological abnormalities could be observed in cells surviving low RH values, but their initial rate of multiplication after transfer to Sabouraud medium was greatly reduced, compared to that demonstrated by cells maintained at 100% RH. At 60% RH, the exposure time required to kill 50% of the blastospores was 2 to 3.5 days. The inimical effect of 60% RH was confirmed in a total of 21 isolates of C. albicans. No deleterious effect was noted when 12 other species of yeasts were subjected to 10, 60, and 100% RH. The single isolate of Candida brumptii and 1 out of the 20 isolates of Cryptococcus neoformans tested also failed to grow after blastospores had been exposed to 60% RH for 4 days.     

Comparison of the Effects of Liquid Medium Repair and the Incorporation of Catalase in MacConkey Type Media on the Recovery of Enterobacteriaceae Sublethally Stressed by Freezing

Nine pure cultures of species of Enterobacteriaceae were stressed by rapid freezing in tryptone soya broth (TSB) to — 22°C and subsequent storage at that temperature for 7 d. About one to two log cycles kill and at least one additional log cycle sublethal impairment was achieved. Numbers of colonies of these cultures in poured plates of violet red bile glucose (VRBG) agar, with 67 u/ml of catalase added at 47°C, were only slightly higher than those in plain VRBG, both incubated overnight at 30°C. Two hours incubation of TSB suspensions at 17–25° C resulted in almost complete restoration of the ability of cells to develop colonies in VRBG, without, however, leading to any significant multiplication.
Similar experiments with 32 samples of frozen minced meat, 27 samples of frozen surface water, 18 of frozen chicken liver and 14 of fresh sausage substantiated the results obtained in the studies on pure cultures.
In the experiments with the nine pure cultures the influence of the nutrient composition of the solid enumeration media: 'minimal' agar, TSB agar (TSBA) and Mueller-Hinton agar with Polyvitex nutrient supplement (MHA), on the recovery of Enterobacteriaceae stressed by freezing was also studied. Colony numbers in TSBA and MHA were virtually identical. The glucose mineral salts medium led to lower recovery, indicating that so-called 'minimal medium recovery' of stressed bacterial populations is not a common phenomenon.     

Pimelic Acid as a Degradation Product of Azelaic Acid by Yeasts

Ebp2p is essential for the assembly of 60S ribosomal subunits, and it interacts with other ribosome assembly factors in Saccharomyces cerevisiae. Two-hybrid screening exhibited that Ebp2p interacted with a small ubiquitin-related modifier (SUMO)-ligase Siz2p and SUMO-related proteins, Ris1p and Wss1p. Mutations of SUMO attachment sites of Ebp2p led to significantly weak interactions with Siz2p, Wss1p, and Ris1p, whereas they exhibited positive interactions with ribosome assembly factors. A SUMO-binding motif of Ris1p was required for interaction with Ebp2p. These results suggest that SUMO mediates the interaction between Ebp2p and SUMO related proteins and that Ebp2p switches its interaction partners via sumoylation.     

Plating of isolated tobacco mesophyll protoplasts on agar medium

Summary A technique was developed to derive cell and plant clones from isolated mesophyll protoplasts of tobacco. The protoplasts, plated on a fully defined agar medium, divided and grew actively forming visible colonies after one month of culture. Efficiency of colony formation depended on cell density and light condition during incubation. Under standard conditions, 60% of plated protoplasts formed colonies. Upon transfer onto suitable media, these colonies differentiated shoots and roots, and eventually regenerated whole plants. Advantages of mesophyll protoplasts as the source of clones as well as implication of the plating technique for genetical studies are discussed.