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Saccharomyces cerevisiae resistance to chlorinated phenoxyacetic acid herbicides involves Pdr1p-mediated transcriptional activation of TPO1 and PDR5 genes.

The transcription regulator Pdr1p is a determinant of Saccharomyces cerevisiae resistance to 2-methyl-4-chlorophenoxyacetic acid (MCPA) and 2,4-dichlorophenoxyacetic acid (2,4-D). The Pdr1p-regulated genes, TPO1 and PDR5, encoding putative multidrug transporters belonging to the major facilitator superfamily (MFS) and to the ATP-binding cassette (ABC) superfamily, respectively, are required for yeast resistance to sudden exposure to these herbicides. A rapid and transient activation of TPO1 (sixfold) and PDR5 (twofold) transcription takes place during the adaptation period preceding cell division under MCPA or 2,4-D moderate stress. These activations are mediated by both Pdr1p and Pdr3p and, as soon as adapted cells start duplication under herbicide stress, mRNA levels are drastically reduced to basal values. The longer duration of the adaptation period, observed for the Delta(pdr1) population, may involve the abolishment of the Pdr1p-mediated transcriptional activation of TPO1 and PDR5 genes, whose expression is critical to surpass the viability loss during the initial period of adaptation to the herbicides.

2,4-Dichlorophenoxyacetic Acid↗

Development of a new oat-based probiotic drink.

In the present work, a whole-grain oat substrate was fermented with lactic acid bacteria to obtain a drink, combining the health benefits of a probiotic culture with the oat prebiotic beta-glucan. The levels of several factors, such as starter culture concentration, oat flour and sucrose content, affecting the fermentation process, were established for completing a controlled fermentation for 8 h. The viable cell counts reached at the end of the process were about 7.5 x 10(10) cfu ml(-1). It was found that the addition of sweeteners aspartame, sodium cyclamate, saccharine and Huxol (12% cyclamate and 1.2% saccharine) had no effect on the dynamics of the fermentation process and on the viability of the starter culture during product storage. Beta-glucan content in the drink (0.31-0.36%) remained unchanged both throughout fermentation and storage of the drink. The shelf life of the oat drink was estimated to 21 days under refrigerated storage.

Avena↗

Effects of aeration and storage temperature on Campylobacter concentrations in poultry semen.

Campylobacter is one of the most commonly reported bacterial causes of human foodborne infections in the United States. Recent evidence has demonstrated that Campylobacter is present in poultry semen and may contribute to the vertical transmission between the breeder hen and offspring. As Campylobacter is considered sensitive to oxygen and cold temperature, the objective of this study was to determine if aeration and storage temperature could reduce or eliminate Campylobacter in poultry semen. In 4 separate trials, pooled semen samples were collected from roosters or toms, diluted with a commercial poultry semen extender, and inoculated with an average of 10(7) cells/mL of a wild-type C. jejuni or C. coli semen isolate. Pooled ejaculates were then divided into 3 aeration treatments: Control (no aeration), air, or oxygen (gently bubbled for 20 min with atmospheric air or oxygen, respectively). Immediately after aeration, pooled semen samples were further divided to 3 test storage temperatures: 4, 23, or 42 degrees C. At 0, 2, 6, and 24 h of storage, samples were enumerated for Campylobacter. Aeration of the semen alone, or aeration with reduced temperatures (4 or 23 degrees C), did not reduce the amount of Campylobacter compared with the controls. Campylobacter concentrations were, however, reduced when stored at 42 degrees C for 24 h. This effect is associated with reduced sperm viability and is therefore not a practical treatment of reducing Campylobacter in semen. It appears alternative methods will be needed to eliminate Campylobacter from poultry semen.

Animals↗

Detection of live and antibiotic-killed bacteria by quantitative real-time PCR of specific fragments of rRNA.

Assessing bacterial viability by molecular markers might help accelerate the measurement of antibiotic-induced killing. This study investigated whether rRNA could be suitable for this purpose. Cultures of penicillin-susceptible and penicillin-tolerant (Tol1 mutant) Streptococcus gordonii were exposed to mechanistically different penicillin and levofloxacin. Bacterial survival was assessed by viable counts and compared to quantitative real-time PCR amplification of either the 16S rRNA genes or the 16S rRNA, following reverse transcription. Penicillin-susceptible S. gordonii lost > or =4 log(10) CFU/ml of viability over 48 h of penicillin treatment. In comparison, the Tol1 mutant lost < or =1 log(10) CFU/ml. Amplification of a 427-bp fragment of 16S rRNA genes yielded amplicons that increased proportionally to viable counts during bacterial growth but did not decrease during drug-induced killing. In contrast, the same 427-bp fragment amplified from 16S rRNA paralleled both bacterial growth and drug-induced killing. It also differentiated between penicillin-induced killing of the parent and the Tol1 mutant (> or =4 log(10) CFU/ml and < or =1 log(10) CFU/ml, respectively) and detected killing by mechanistically unrelated levofloxacin. Since large fragments of polynucleotides might be degraded faster than smaller fragments, the experiments were repeated by amplifying a 119-bp region internal to the original 427-bp fragment. The amount of 119-bp amplicons increased proportionally to viability during growth but remained stable during drug treatment. Thus, 16S rRNA was a marker of antibiotic-induced killing, but the size of the amplified fragment was critical for differentiation between live and dead bacteria.

Anti-Bacterial Agents↗

Chronic posttraumatic osteomyelitis and infected nonunion of the tibia: current management concepts.

Chronic posttraumatic osteomyelitis and infected nonunion of the tibia are complex problems that result in considerable morbidity and can threaten viability of the limb. Development of infection may result from compromised soft tissue and bone vascularity, systemic compromise of the host, and virulent or resistant organisms. Biofilm formation on implant and devascularized bone surfaces protects pathogens and may lead to persistence of infection. Management is based on a detailed evaluation of the patient, the involved bone and soft tissues, degree of associated lower extremity injury, and type of bacterial pathogens. Infection control is achieved with radical débridement, skeletal stabilization, and microbial-specific antibiotics. Local antibiotic delivery is a useful supplement to systemic administration. Local or free muscle flaps may be necessary to achieve soft-tissue coverage. Restoration of bone defects and bony union can be accomplished with bone grafting. However, large defects require complex reconstructive procedures, such as distraction osteogenesis and vascularized bone grafting.

Chronic Disease↗

[Replicative properties of HIV-1 strains, resistant to 3'-azido-2'3'-dideoxythymidine].

The replicative activity of two strains of human immunodeficiency virus type I, 1974 and 1978, resistant to 3'-azido-2',3'-dideoxythymidine (AZT), was investigated. Assessment of AZT sensitivity by the standard method showed the 50% effective dose (ED50) for strain 1974 to be 0.1 mumole and for strain 1978, 1 mumole. Both strains had the slow/low. 3 phenotype. The level of virus-specific antigens in these strains was 3-7 times lower than in AZT-sensitive strain IIIB. In order to study the effects of AZT on virus replication, the strains were passaged in Jurkat-tat cells in the presence of 3.5 mumole of AZT. Virus activity was assessed from the level of intracellular (S-12) and extracellular (P-100) antigens, cell viability, and syncytium formation. Cell viability increased by 10%, syncytium formation dropped 5 times, and antigen accumulation increased 1.5-2 times in the culture with strain 1974. For strain 1978 these parameters were as follows: 17% decrease of cell viability, increase of syncytium formation by 1.7 times, and no changes in the level of viral antigen. After four serial passages in the presence of AZT ED50 for strain 1974 was 0.5 mumole and at least 1 mumole for strain 1978.

Cell Survival↗

A Comparison of the Survival of Intraperiplasmic and Attack Phase Bdellovibrios with Reduced Oxygen

The ability of intraperiplasmic and attack phase bdellovibrios to survive and/or grow under anoxic and microaerobic conditions was examined. Both halotolerant and nonhalotolerant bdellovibrio strains were examined. In all instances, the bdellovibrio strains were unable to grow under anoxic conditions, but were able to survive for periods of time in both the extracellular and intraperiplasmic forms. However, the intraperiplasmic organisms were observed to survive longer. Increased temperature hastened the loss of viability of both forms of the predatory bacteria in oxic and anoxic environments. Under microaerobic conditions, halotolerant bdellovibrios were observed to grow, although at a slightly reduced rate than in atmospheric oxygen, while two nonhalotolerant isolates survived but did not grow. The ability of attack phase bdellovibrios to survive in an anoxic environment for up to nine days and their growth or survival under microaerobic conditions greatly expands the possible ecological niches in which the predators may be active members of the microbial community.

Journal Article↗

Microorganisms as tracers in groundwater injection and recovery experiments: a review.

Modern day injection and recovery techniques designed to examine the transport behavior of microorganisms in groundwater have evolved from experiments conducted in the late 1800s, in which bacteria that form red or yellow pigments were used to trace flow paths through karst and fractured-rock aquifers. A number of subsequent groundwater hydrology studies employed bacteriophage that can be injected into aquifers at very high concentrations (e.g., 10(13) phage ml-1) and monitored through many log units of dilution to follow groundwater flow paths for great distances, particularly in karst terrain. Starting in the 1930s, microbial indicators of fecal contamination (particularly coliform bacteria and their coliphages) were employed as tracers to determine potential migration of pathogens in groundwater. Several injection and recovery experiments performed in the 1990s employed indigenous groundwater microorganisms (both cultured and uncultured) that are better able to survive under in situ conditions. Better methods for labeling native bacteria (e.g. by stable isotope labeling or inserting genetic markers, such as the ability to cause ice nucleation) are being developed that will not compromise the organisms' viability during the experimental time course.

Enterobacteriaceae↗

Effect of high hydrostatic pressure on Cryptosporidium parvum infectivity.

The incidence of foodborne disease outbreaks caused by contaminated low-pH fruit juices is increasing. With recent mandatory pasteurization of apple juice and the industry's concerns of food safety, fruit juice processors are showing more interest in alternative nonthermal technologies that can kill >99.99% of microbial pathogens present in foods. The association of the coccidian protozoan, Cryptosporidium, with diarrheal disease outbreaks from contaminated tap water and fruit juice raises a safety concern in the food and beverage industries. The objective of this study was to evaluate the effects of high hydrostatic pressure (HHP) on C. parvum oocysts. Oocysts were suspended in apple and orange juice and HHP treated at 5.5 x 10(8) Pa (80,000 psi) for 0, 30, 45, 60, 90, and 120 s. Oocyst viability was assessed by excystation using bile salts and trypsin while the cell culture foci detection method was used to assess infectivity. Results indicated that HHP inactivated C. parvum oocysts by at least 3.4 log10 after 30 s of treatment. No infectivity was detected in samples exposed to > or =60 s of HHP and >99.995% inactivation was observed. This study demonstrated that HHP efficiently rendered the oocysts nonviable and noninfectious after treatment at 5.5 x 10(8) Pa.

Animals↗

Cell elongation and cell death of helicobacter pylori is modulated by the disruption of cdrA (cell division-related gene A).

The cell division-related gene A (cdrA) of Helicobacter pylori is dispensable in vivo and unique in having a repressive role on cell division and long-term survival. To clarify its role, comparisons of the wildtype HPK5 and isogenic cdrA-disrupted mutant HPKT510 were examined by ultrastructural morphology, PBP profiles, and susceptibility to beta-lactam antibiotics during long-term cultivation. Ultrastructural analyses revealed that the shorter rods of HPKT510 had a slightly wider periplasmic space between the inner and the outer membrane than those of HPK5. Cell division of HPKT510 cells was complete even under high-salt conditions in which HPK5 cells became filamentous due to inhibition of division. The filamentous HPK5 cells constructed an inner membrane without a cell wall at the presumed division site. After 4 days of cultivation (the late stationary phase), most of the HPK5 cells turned into ghosts and aggregates, while some of the HPKT510 cells remained as curved rods, which coincided with the results of cell viability. HPKT510 cells became resistant to ampicillin killing compared to HPK5 cells, although their minimum inhibitory concentrations (MICs) and PBP profiles were not significantly different. These results suggest that the cdrA product represses cell division via inhibiting cell wall synthesis at division site. During infection in both mice and humans, inactivation of cdrA eventually gains biological aspects such as increased viability, long-term survival and tolerance to antibiotics and high-salt condition, which might enhance a persistent infection.

Anti-Bacterial Agents↗

Candidicidal activity of the human recombinant myeloperoxidase system.

The effect of the human recombinant myeloperoxidase (recMPO) system on the viability of Candida albicans blastospores was determined. Cells were incubated in the presence of the enzyme, H2O2, and various substrates. In optimal conditions, 100% mortality was reached with only 41 mU of recMPO per ml, confirming its powerful candidicidal activity.

Candida albicans↗

Development of a probiotic cheddar cheese containing human-derived Lactobacillus paracasei strains.

Cheddar cheese was manufactured with either Lactobacillus salivarius NFBC 310, NFBC 321, or NFBC 348 or L. paracasei NFBC 338 or NFBC 364 as the dairy starter adjunct. These five strains had previously been isolated from the human small intestine and have been characterized extensively with respect to their probiotic potential. Enumeration of these strains in mature Cheddar cheese, however, was complicated by the presence of high numbers (>10(7) CFU/g of cheese) of nonstarter lactic acid bacteria, principally composed of lactobacilli which proliferate as the cheese ripens. Attempts to differentiate the adjunct lactobacilli from the nonstarter lactobacilli based on bile tolerance and growth temperature were unsuccessful. In contrast, the randomly amplified polymorphic DNA method allowed the generation of discrete DNA fingerprints for each strain which were clearly distinguishable from those generated from the natural flora of the cheeses. Using this approach, it was found that both L. paracasei strains grew and sustained high viability in cheese during ripening, while each of the L. salivarius species declined over the ripening period. These data demonstrate that Cheddar cheese can be an effective vehicle for delivery of some probiotic organisms to the consumer.

Amino Acids↗

Inoculation of BALB/c mice with Lacazia loboi.

In a previous study, the authors inoculated Swiss mice with Lacazia loboi (L. loboi) and succeeded in maintaining a granulomatous infiltrate and viable fungal cells up to one year and six months after inoculation. Considering the experimental work on paracoccidioidomycosis, 0.03 ml of a fungal suspension obtained from a biopsy of a Jorge Lobo's Disease patient were inoculated into both hind foot pads of 32 six week-old BALB/c mice of both sexes. The animals were sacrificed 1, 4, 7 and 10 months post inoculation. The suspension contained 1.3 x 10(6) fungi/ml and presented 38% viability. Seven months after inoculation, most of the animals presented profuse infiltrates consisting of isolated histiocytes, foreign body and Langhans' giant cells and a large number of fungi, most of them viable. Emergence of macroscopic lesions was observed during the 8th month. Based on fungal count, viability index before and after inoculation, presence of macroscopic lesions and histopathological findings similar to the findings in humans, the authors believe that BALB/c mice may be a good experimental model to study Jorge Lobo's Disease, mainly regarding therapeutic evaluation.

Animals↗

Lactobacillus rhamnosus strain GG is a potential probiotic for calves.

Diarrhea is a common occurrence in neonatal calves. Several veterinary probiotics claiming to prevent or treat calf diarrhea are available, but have not been well studied. This study assessed the capability of Lactobacillus rhamnosus strain GG (LGG) to maintain viability in the gastrointestinal tract of calves. We also determined whether LGG can be administered in an oral rehydration solution (ORS) without compromising the efficacy of the ORS or the viability of LGG, and whether LGG produces D-lactate or not. To investigate the intestinal survival of LGG, 15 calves were randomized into 3 groups and LGG was administered orally with their morning milk feeding on 3 consecutive days at a low (LD), medium (MD), or high (HD) dosage. Fecal samples were collected on days 0 (control), 1, 2, 3, 5, and 7 and incubated for 72 h on deMan, Rogosa, Sharpe agar. Twenty-four hours after the 1st feeding, LGG was recovered from 1 out of 5 calves in the LD group, 4 out of 5 calves in the MD group, and 5 out of 5 calves in the HD group. To determine if LGG caused the glucose levels in the ORS to drop below effective levels, 1.5 L of the ORS was incubated with LGG for 2 h at 37 degrees C and the glucose concentration was measured every 20 min using a glucose meter. This ORS was then further incubated for 10 h and aliquots analyzed by high performance liquid chromatography to determine if D-lactate was produced by LGG. Glucose concentrations did not change over the 2 h of incubation, and no D-lactate was produced after 48 h. The LGG maintained viability in ORS. Therefore, this study demonstrated that LGG survives intestinal transit in the young calf, produces no D-lactate, and can be administered in an ORS.

Administration, Oral↗

The bactericidal activity of vancomycin and teicoplanin against Streptococcus pneumoniae.

The in vitro inhibitory and bactericidal activity of vancomycin and teicoplanin against clinical isolates of Streptococcus pneumoniae was investigated. Minimum inhibitory concentrations (MICs) were determined using an agar incorporation technique. Vancomycin (MIC range 0.12-0.25 mg/l) and teicoplanin (MIC range 0.03-0.12 mg/l) were both very active against the isolates examined. Single-time-point minimum bactericidal concentrations (MBCs) were determined using a macrobroth incorporation technique. Both antimicrobials brought about a 99.9% reduction in viability of the original inocula during 24 hours' exposure at concentrations of 4-8 times the MIC for vancomycin and 2-4 times the MIC for teicoplanin. Time-kill curves of vancomycin, teicoplanin and penicillin against a penicillin-susceptible and a penicillin-resistant strain of S. pneumoniae showed a greater than 3.0 log10 cfu/ml reduction in viability of cultures within 6 hours of exposure to all three of the antimicrobials at a concentration equal to eight times the MIC. The results suggest that clinical trials of the efficacy of teicoplanin in the treatment of infections caused by S. pneumoniae are warranted.

Glycopeptides↗

Sterility and use patterns of multiple-dose vials.

The viability of microorganisms in multiple-dose vials (MDVs) and the use and in-use contamination rate of MDVs were investigated. Serial tenfold dilutions of stationary cultures of Escherichia coli and Pseudomonas aeruginosa were injected into 30-ml MDVs containing bacteriostatic agents, and samples were removed at 1, 16, 24, and 48 hours, and at seven days to test for viable organisms. All opened MDVs were removed from each patient-care area and the pharmacy in a hospital and tested for microbial contamination using an aliquot-sampling method. One nursing unit was visited each day, in random order, until all opened MDVs from all units and the pharmacy were collected. The day following collection, all newly opened MDVs at each unit were marked inconspicuously and tallied. On the first, sixth, and thirteenth day after marking, all marked MDVs remaining on the unit were tallied. Bacteria were isolated from deliberately contaminated MDVs when inoculated with 1-100 colony-forming units/ml or greater when the sample was tested within one hour after contamination. Only one product was positive in 16 hours, and none was positive beyond that time. A total of 928 opened MDVs was collected from 31 nursing units and the pharmacy; none was positive for microbial contamination, indicating that the contamination rate was probably less than 4 per 1000. Lidocaine, insulin, diluents, and heparin constituted 57% of collected vials. The length of time that opened vials remained on a unit and the number of opened vials per unit varied considerably between units. The cost, feasibility, and effectiveness of control policies regarding use of MDVs should be weighed objectively against potential benefits.

Drug Contamination↗

An economic and technical assessment of the use of plant cell cultures for natural product synthesis on an industrial scale.

Plant cell cultures may be used as an alternative source of established natural products, as a source of novel 'lead' compounds or as a source of enzymes for modification of precursors. Only a few plant cell processes are operating commercially and their performance characteristics are industrial secrets. The economic aspects of natural product synthesis in plant cell cultures are presented on the basis of data derived from work on a pilot plant with bioreactors of 5-80 litres in which cells are grown in batch liquid culture. Cost analysis shows that the labour costs of operating plant cell culture processes are much higher than those for microbial processes, which reflects the longer process times of plant systems. These can be reduced by increasing the cell growth rate, the biomass yield and/or the product yield. Higher yields can be obtained by optimizing media conditions, but there are no standard guidelines for this. Each system has to be developed individually. Reducing the number of production runs a year, usually by increasing the number of days for which each batch of cells is synthesizing product, can markedly decrease costs. Economic assessment of the viability of production in plant cell cultures must consider not only production costs but also the expected market price of the product and the volume of sales.

Biological Products↗

Genetic damage during thymidylate starvation in Saccharomyces cerevisiae.

Thymidylate starvation in a yeast mutant auxotrophic for dTMP caused cell death and the induction of mutations in the mitochondrial genome. After 24 h of starvation almost all surviving cells were respiratory deficient petites. In addition, shorter episodes of dTMP starvation induced chloramphenicol and erythromycin resistant mutants, indicating the occurrence of mitochondrial point mutations. Suboptimal concentrations of exogenous thymidylate were also found to induce petites and a decline in cell viability and the magnitude of these effects was acutely dependent upon the dTMP concentration. Cesium chloride gradient analysis of DNA from cells undergoing thymineless incubation revealed a progressive loss of mitochondrial DNA, and a decrease in the molecular weight of nuclear DNA.

Chloramphenicol↗