Activities of Linezolid against nontuberculous mycobacteria

The activity of linezolid (Pfizer, USA) was tested by broth microdilution against 53 clinical isolates of non-tuberculous mycobacteria (NTM), including the common disease producing species Mycobacterium avium, M. intracellulare, M. fortuitum, M. chelonae and M. abscessus, obtained from western Turkey. The isolates of M. abscessus and M. intracellulare were the least susceptible, M. mucogenicum, M. gordonae and M. avium were the most susceptible to linezolid of the common species of NTM. Linezolid showed a variable sensitivity in all strains; therefore, each species and strain must be individually evaluated, and it is always advisable to perform in vitro sensitivity tests before using the drug for human therapy.     

Incidence of nontuberculous mycobacteria in four hot water systems using various types of disinfection

The objective of this study was to determine the incidence of nontuberculous mycobacteria (NTM) in hot water systems of 4 selected hospital settings. The hospitals provided the following types of disinfection for their hot water systems: hydrogen peroxide and silver, thermal disinfection, chlorine dioxide, and no treatment (control). In each building, 6 samples were collected from 5 sites during a 3 month period. NTM were detected in 56 (46.7%) of 120 samples; the CFU counts ranged from 10 to 1625 CFU/L. The detected NTM species were the pathogens Mycobacterium kansasii, Mycobacterium xenopi, and Mycobacterium fortuitum and the saprophyte Mycobacterium gordonae. The most common to be isolated was M. xenopi, which was present in 51 samples. The hot water systems differed significantly in the incidence of NTM. NTM were not detected in the system treated by thermal disinfection, and a relatively low incidence (20% positive samples) was found in the system disinfected with chlorine dioxide. However, a high incidence was found in the control system with no additional disinfection (70% positives) and in the system using hydrogen peroxide and silver (97% positives). Water temperatures above 50 degrees C significantly limited the occurrence of NTM.     

Variability of Bacillus thuringiensis under various growth conditions

When a lysogenic culture of Bacillus thuringiensis subsp. galleriae 69-6 was grown under the batch conditions, 93-99% of cells in the population produced R-form colonies and ca. 1% yielded S-form colonies. The amount of spore-forming cells was 99% in R-variants and 8% in S-variants. The quantity of S-variants rose abruptly to 99% when the culture was grown under the chemostat conditions. The number of S-variants increased with the rate and the duration of growth. The process was influenced by growth-limiting factors. Temperate phage variants capable of host culture lysis on solid media (i.e. h-mutants) were not found under the conditions of batch cultivation. However, such phage particles (h-mutants) appeared under the conditions of chemostat. The titre of these phage particles reached 10(8), 10(7) and 10(4) particles per 1 ml at limitation with yeast extract, glucose and phosphorus, respectively. Under the conditions of chemostat, the particles behaved as temperate ones and their growth was not found. Irrespective of the limitation, the phage titre did not correlate with the ratio of R and S-forms in the population. When the growth was limited with phosphorus, the quantity of S-forms increased abruptly while the spontaneous induction of the phage was inhibited. The quantity of cells capable of spore formation decreased in the cultures isolated from the chemostat and grown on MPA: 69-80% of the cells in R-forms and merely 8% in S-forms.     

Occurrence of nontuberculous mycobacteria in environmental samples.

Nontuberculous mycobacteria (NTM) are a major cause of opportunistic infection in immunocompromised hosts. Because there is no evidence of person-to-person transmission and NTM have been found in drinking water, the environment is considered a likely source of infection. In this study the widespread occurrence of NTM was examined in drinking water, bottled water, and ice samples. A total of 139 samples were examined for NTM by a membrane filtration culture technique followed by PCR amplification and 16S rRNA sequence determination to identify the isolates. NTM were not detected in bottled water or cisterns but were detected in 54% of the ice samples and 35% of the public drinking-water samples from 21 states. The most frequently occurring isolate was M. mucogenicum (formerly referred to as an M. chelonae-like organism).     

Shifting paradigms of nontuberculous mycobacteria in cystic fibrosis

Important paradigms of pulmonary disease with nontuberculous mycobacteria (NTM) are currently shifting based on an increasing attention within the field of cystic fibrosis (CF). These shifts are likely to benefit the management of all patients with pulmonary NTM, regardless of underlying pathology. Currently several key areas are being revised: The first outbreak of human NTM transmission has been proven and new evidence of biofilm growth in vivo has been demonstrated. A better understanding of the clinical impact of NTM infection has led to increased diagnostic vigilance and new recommendations for lung transplantation are under way. While recent changes have reinvigorated the interest in NTM disease, the challenge remains, whether such advances can be successfully translated into improved management and care.     

Accumulation of polyhydroxyalkanoates by Microlunatus phosphovorus under various growth conditions

Microlunatus phosphovorus is an activated-sludge bacterium with high levels of phosphorus-accumulating activity and phosphate uptake and release activities. Thus, it is an interesting model organism to study biological phosphorus removal. However, there are no studies demonstrating the polyhydroxyalkanoate (PHA) storage capability of M. phosphovorus, which is surprising for a polyphosphate-accumulating organism. This study investigates in detail the PHA storage behavior of M. phosphovorus under different growth conditions and using different carbon sources. Pure culture studies in batch-growth systems were conducted in shake-flasks and in a bioreactor, using chemically defined growth media with glucose as the sole carbon source. A batch-growth system with anaerobic–aerobic cycles and varying concentrations of glucose or acetate as the sole carbon source, similar to enhanced biological phosphorus removal processes, was also employed. The results of this study demonstrate for the first time that M. phosphovorus produces significant amounts of PHAs under various growth conditions and with different carbon sources. When the PHA productions of all cultivations were compared, poly(3-hydroxybutyrate) (PHB), the major PHA polymer, was produced at about 20–30% of the cellular dry weight. The highest PHB production was observed as 1,421 mg/l in batch-growth systems with anaerobic–aerobic cycles and at 4 g/l initial glucose concentration. In light of these key results regarding the growth physiology and PHA-production capability of M. phosphovorus, it can be concluded that this organism could be a good candidate for microbial PHA production because of its advantages of easy growth, high biomass and PHB yield on substrate and no significant production of fermentative byproducts.     

Differential expression of Arabidopsis sulfurtransferases under various growth conditions.

Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyse the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. Neither the in vivo sulfur donors nor the acceptors of Str could be clearly identified in any of the organisms investigated so far. In Arabidopsis thaliana 20 Str proteins have been identified and grouped according to sequence homology. To investigate their respective in vivo function, Arabidopsis plants were grown in sterile hydroponic cultures at different sulfate (50, 500, and 1500 microM) and phosphate (0.1 and 1mM) concentrations, and in medium supplemented with 1mM thiosulfate. Northern blot analysis revealed the differential expression of the Str investigated. Thiosulfate Str activity was significantly increased at low sulfate concentrations in the medium. The Str mRNA levels were highly dependent on the developmental stage of the Arabidopsis plants. The expression of most Str analysed increased with progressing plant age in parallel with increasing 3-mercaptopyruvate and thiosulfate Str activities. The Str investigated were differentially expressed in a light/dark cycle whereas Str enzyme activities were not affected by the light conditions. The results indicate that each Str is regulated in a different way and plays an individual specific role in the plant metabolism.     

L-asparaginase activity of Mycobacterium phlei under various growth conditions.

Seven Mycobacterium strains were grown statically on salts-glycerol-asparagine (Sauton) or on salts-glucose-glutamate (Sym) media. At desired time of incubation, the bacteria were washed with water, disintegrated with powdered corundum and in resulting cell-free extracts L-asparaginase activity was determined by the Conway method. The majority of experiments were performed on M. phlei which exhibited considerable rise in L-asparaginase activity with increasing age of the culture. This change did not occur on Sym medium because of Zn2+, which proved to abolish the effect of the enzyme induction in vivo but did not inhibit the activity in vitro. Addition of rifampicin to Sauton culture media resulted in a low enzyme level. Exogenous asparagine and glycerol were not indispensable for the enzyme synthesis and could be replaced by glutamate and glucose, respectively.     

Marine phytoplankton growth and survival under simulated upwelling and oligotrophic conditions

Dialysis cultures of a diatom, Chaetoceros affinis Lauder, and a dinoflagellate, Gymnodnium splendens Lebour were subjected to simulated upwelling conditions which were followed by oligotrophy. Upwelling was simulated by pumping nutrient-enriched sea water past the enclosed cultures. Oligotrophy was simulated by pumping nutrient-poor sea water past the cultures. After the period of upwelling oligotrophy was carried on for 65 days for G. splendens and 80 days for C. affinis. Both algae survived oligotrophic conditions as evidenced by the ability to initiate new cultures or by observation of motility. Nutrient analyses showed that oligotrophy developed slowly — over a period of several days — so that the cultures were subjected to slow changes similar to those which might occur in the sea when upwelling ceases or when upwelled water moves offshore. C. affinis responded to oligotrophy by developing small flagellated cells (male gametes), by losing discrete protoplasts in silicified cells, and by the breaking up of normal chains of cells. Plastids in G. splendens cells became more discrete under oligotrophic conditions and some large non-flagellated cells were observed. The use of dialysis cultures to simulate features of the ocean is discussed.     

Identification of nontuberculous mycobacteria by methods of molecular biology

A number of clinical and laboratory strains of microorganisms belonging to different species of mycobacteria of the nontuberculous complex were tested with the use of the polymerase chain reaction and the restriction analysis. The unique restriction profiles of the following species of mycobacteria have been obtained: M. fortuitum VI, M. kansasii I, M. intracellulare, M. avium.     

Automated analysis and survival selection of anchorage-dependent cells under normal growth conditions

An instrument is described that can automatically analyze and select for a subpopulation of anchorage-dependent cells in tissue culture. Cells that label with fluorescently tagged antibodies or demonstrate structural variations are saved from exposure to a destructive high-intensity argon laser beam. The surviving population may then be cloned. The cell selection may occur in a tissue culture plate or in a microflow incubator which is designed to maintain a constant flow of media at 37 degrees C across cells growing on a glass coverslip. This incubator sits on an inverted microscope which focuses the laser beam to a diameter as small as 1 micron. A high-speed computer-controlled two-dimensional stage moves the cells past the beam for analysis, the results of which determine the fate of each cell: whether it is to be destroyed by radiant energy or selected for survival and subsequent proliferation. Another selection strategy performed by the instrument involves growing the cells on a thin, blackened polyester film which can be cut by the argon laser beam. Cells selected for cloning are then circumscribed. The heat of cutting welds the circumscribed film to a plastic coverslip surface or tissue culture chamber bottom. Nonselected cells may be removed by pulling the unattached polyester sheet from the attachment surface. The selected cells remain on polyester film disks welded to the plastic. Selections may be done automatically under computer control or manually by operator direction of stage movements. This instrument extends the art of automated cell selection and analysis to normal cell lines that must maintain cell-substratum contact (anchorage dependence) for differentiated cell function, e.g., neurons, fibroblasts, or kidney cells.     

Survival of directionally solidified B-lymphoblasts under various crystal growth conditions.

Reduction of temperature during freezing brings about two complex and interrelated phenomena: (1) crystal nucleation and subsequent growth processes and (2) change in biophysical properties of a biological system. The purpose of this investigation is to relate the morphology of the solid phase with the survival of a cell. To this end, B-lymphoblasts were exposed to directional solidification in phosphate-buffered saline + 0.05 M dimethyl sulfoxide. Directional solidification is a freezing technique which allows the morphology of the interface to be varied without varying the chemical history that a cell would experience during a constant cooling rate protocol. Results indicated that, for the range of experimental conditions tested, a maximum survival of approximately 78% could be achieved using a temperature gradient of 25(10)3 K/m and an interface velocity of 23(10)-6 m/s (cooling rate: 35 K/min). Survival dropped off sharply for freezing at faster cooling rates with little or no variation in survival for different crystal growth conditions. Survival at slower cooling rates decreased with decreasing cooling rate. It was observed, however, that the presence of secondary branches in the ice phase correlated with lower survival for a given cooling rate. These results indicated that not only is the redistribution of solute during freezing a potential source of damage during freezing but ice/cell interactions are also. Thus, the cooling rate alone may not be adequate to describe the freezing process.     

Phenotypic heterogeneity as key factor for growth and survival under oligotrophic conditions

Productivity-poor oligotrophic environments are plentiful on earth. Yet it is not well understood how organisms maintain population sizes under these extreme conditions. Most scenarios consider the adaptation of a single microorganism (isogenic) at the cellular level, which increases their fitness in such an environment. However, in oligotrophic environments, the adaptation of microorganisms at population level – that is, the ability of living cells to differentiate into subtypes with specialized attributes leading to the coexistence of different phenotypes in isogenic populations – remains a little-explored area of microbiology research. In this study, we performed experiments to demonstrate that an isogenic population differentiated to two subpopulations under low energy-flux in chemostats. Fluorescence cytometry and turnover rates revealed that these subpopulations differ in their nucleic acid content and metabolic activity. A mechanistic modelling framework for the dynamic adaptation of microorganisms with the consideration of their ability to switch between different phenotypes was experimentally calibrated and validated. Simulation of hypothetical scenarios suggests that responsive diversification upon a change in energy availability offers a competitive advantage over homogenous adaptation for maintaining viability and metabolic activity with time.     

Prevalence of nontuberculous mycobacteria in water supplies of hemodialysis centers

Infection of hemodialysis patients with nontuberculous mycobacteria (NTM) has been associated with water used in reprocessing hemodialyzers. This study was conducted to determine the prevalence of NTM and other bacteria in water samples collected over a 13-week period from 115 randomly selected dialysis centers in the United States. Total viable counts were determined by membrane filter assays; increased recovery of NTM was obtained by dosing a portion of each water sample with 1% formaldehyde (HCHO) before filtering. NTM were widely distributed and occurred with a high frequency in water supplies in dialysis centers. NTM were detected in water from 95 centers (83%), and 50% of all samples examined contained NTM. The results of this study support recommendations to use 4% HCHO or a chemical germicidal equivalent for disinfecting dialyzers that are to be reused.     

Macromolecule synthesis in Escherichia coli BB under various growth conditions.

The kinetic behavior of the macromolecule synthesis of Escherichia coli during balanced growth in various media at different temperatures as investigated. The results indicate that macromolecule contents per cell can be expressed as exponential functions of the specific growth rate at a given temperature. It was shown that the content per cell at the zero growth rate was constant in each macromolecule component, irrespective of the growth temperature. The rate of ribonucleic acid (RNA) synthesis per unit weight of deoxyribonucleic acid and that of protein synthesis per unit weight of RNA were taken as efficiencies of RNA and protein synthesis, respectively; both of them were found to be dependent on the growth rate and temperature. The efficiency of RNA synthesis was found to be very high at a high growth rate, whereas that of protein synthesis was found to decrease above certain growth rate. At the same growth rate, an increase in the growth temperature resulted in a decrease in the efficiency of RNA synthesis but an increase in that of protein synthesis.     

Comparison of some decontamination methods and growth media for isolation of mycobacteria from northern brook waters

Two decontamination methods and five media were compared for the isolation of mycobacteria from brook waters of different physical, chemical and bacteriological characteristics. The decontaminants used were: 0.7 mol 1^-1 NaOH followed by 50 g 1^-1 oxalic acid and 0.9 mol 1^-1 H₂SO₄ combined with 0.5 g 1^-1 cycloheximide. The media compared were: Mycobacteria 7H11 agar with OADC enrichment (pH 6.6), glycerol egg (pH 6.5 and 5.5), and pyruvate egg (pH 6.5 and 5.5). All media contained cycloheximide, 0.5 g 1^-1. The NaOH—oxalic acid method generally resulted in lower contamination and higher isolation of mycobacteria than the H₂SO₄-cycloheximide method. With the NaOH—oxalic acid method, all five media were equal in positivity rates but contamination was a problem on Mycobacteria 7H11 agar. Of the four egg media tested, the highest positivity rate (92% of the samples) was obtained on the pyruvate modification (pH 6.5), and the highest mean colony count of mycobacteria (900 cfu 1^-1) on the glycerol modification (pH 6.5). Characteristics of water and sampling site had similar effects on the isolation frequencies of mycobacteria obtained by different combinations.     

Content and composition of hopanoids in Zymomonas mobilis under various growth conditions.

By using a new method for quantification of the different hopanoid derivatives, a total hopanoid content of about 30 mg/g (dry cell weight) was observed in Zymomonas mobilis. This value is the highest reported for bacteria so far. The major hopanoids in Z. mobilis were the ether and glycosidic derivatives of tetrahydroxy-bacteriohopane, constituting about 41 and 49% of the total hopanoids. Tetrahydroxybacteriohopane itself, diplopterol, and hopene made up about 6, 3, and 1%, respectively. Only minor changes in hopanoid composition were observed with changes in growth conditions. Earlier reports on a correlation between hopanoid content and ethanol concentration in the medium could not be confirmed. Over a wide range of ethanol concentrations (5 to 60 g/liter), growth rates (0.08 to 0.25 h-1), and temperatures (25 to 37 degrees C), the molar ratio of hopanoids to phospholipids in the cells amounted to about 0.7. Only at growth rates of greater than 0.30 h-1 did the molar ratio increase to about 1.