Thomas Egli

Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate

Polyhydroxyalkanoates (PHAs) comprise a large class of polyesters that are synthesized by many bacteria as an intracellular carbon and energy compound. Analysis of isolated PHAs reveal interesting properties such as biodegradability and biocompatibility. Research was focused only recently on the application of PHA in implants, scaffolds in tissue engineering, or as drug carriers. Such applications require that PHA be produced at a constant and reproducible quality. To date this can be achieved best through bacterial production in continuous culture where growth conditions are kept constant (chemostat). Recently, it was found that PHA producing bacteria are able to grow simultaneously limited by carbon and nitrogen substrates. Thus, it became possible to produce PHA at high yields on toxic substrate and also control its composition accurately (tailor-made synthesis). Finally, applications of PHA in medicine are discussed.

Assessment and Interpretation of Bacterial Viability by Using the LIVE/DEAD BacLight Kit in Combination with Flow Cytometry

ABSTRACT The commercially available LIVE/DEAD BacLight kit is enjoying increased popularity among researchers in various fields of microbiology. Its use in combination with flow cytometry brought up new questions about how to interpret LIVE/DEAD staining results. Intermediate states, normally difficult to detect with epifluorescence microscopy, are a common phenomenon when the assay is used in flow cytometry and still lack rationale. It is shown here that the application of propidium iodide in combination with a green fluorescent total nucleic acid stain on UVA-irradiated cells of Escherichia coli, Salmonella enterica serovar Typhimurium, Shigella flexneri, and a community of freshwater bacteria resulted in a clear and distinctive flow cytometric staining pattern. In the gram-negative bacterium E. coli as well as in the two enteric pathogens, the pattern can be related to the presence of intermediate cellular states characterized by the degree of damage afflicted specifically on the bacterial outer membrane. This hypothesis is supported by the fact that EDTA-treated nonirradiated cells exhibit the same staining properties. On the contrary, this pattern was not observed in gram-positive Enterococcus faecalis, which lacks an outer membrane. Our observations add a new aspect to the LIVE/DEAD stain, which so far was believed to be dependent only on cytoplasmic membrane permeability.

The Ecological and Physiological Significance of the Growth of Heterotrophic Microorganisms with Mixtures of Substrates

It has been estimated that globally some 500 × 1012 kg of carbon dioxide are assimilated into biomass by autotrophic organisms annually. More than 99% of this assimilated carbon is remineralized, keeping the global biogeochemical carbon cycle roughly in balance (Hedges, 1992). In both terrestrial and aquatic ecosystems the majority of this primary biomass is not consumed directly by herbivorous animals, but decays to detritus and serves as a nutritional basis for the growth of consumers (for an extensive discussion, see Fenchel and Jorgensen, 1977). There is now substantial evidence suggesting that a large part of the energy and nutrients contained in this primary biomass is processed via the microbial detritus food chain, and this mineralizing ability makes heterotrophic microorganisms an important link in the global carbon cycle (Fenchel and Jorgensen, 1977; Paul and Voroney, 1980; Wetzel, 1984; Cole et al., 1988; Mann, 1988). In addition, their ability to mineralize man-made xenobiotic organic chemicals has become increasingly important. This is illustrated by the fact that in industrialized countries the flux of synthetically produced organic material, much of which is ending up in the environment, has increased within the past two centuries to some 40 g C m−2 year−1. This figure is equivalent to approximately 15% of the net primary biomass production in these regions (Egli, 1992).

Rapid, cultivation-independent assessment of microbial viability in drinking water

Fast and accurate monitoring of chemical and microbiological parameters in drinking water is essential to safeguard the consumer and to improve the understanding of treatment and distribution systems. However, most water utilities and drinking water guidelines still rely solely on time-requiring heterotrophic plate counts (HPC) and plating for faecal indicator bacteria as regular microbiological control parameters. The recent development of relative simple bench-top flow cytometers has made rapid and quantitative analysis of cultivation-independent microbial parameters more feasible than ever before. Here we present a study using a combination of cultivation-independent methods including fluorescence staining (for membrane integrity, membrane potential and esterase activity) combined with flow cytometry and total adenosine tri-phosphate (ATP) measurements, to assess microbial viability in drinking water. We have applied the methods to different drinking water samples including non-chlorinated household tap water, untreated natural spring water, and commercially available bottled water. We conclude that the esterase-positive cell fraction, the total ATP values and the high nucleic acid (HNA) bacterial fraction (from SYBR Green I staining) were most representative of the active/viable population in all of the water samples. These rapid methods present an alternative way to assess the general microbial quality of drinking water as well as specific events that can occur during treatment and distribution, with equal application possibilities in research and routine analysis.

Efficacy of solar disinfection of Escherichia coli, Shigella flexneri, Salmonella Typhimurium and Vibrio cholerae

Aims:  To determine the efficacy of solar disinfection (SODIS) for enteric pathogens and to test applicability of the reciprocity law.

Specific Growth Rate Determines the Sensitivity of Escherichia coli to Thermal, UVA, and Solar Disinfection

ABSTRACT Knowledge about the sensitivity of the test organism is essential for the evaluation of any disinfection method. In this work we show that sensitivity of Escherichia coli MG1655 to three physical stresses (mild heat, UVA light, and sunlight) that are relevant in the disinfection of drinking water with solar radiation is determined by the specific growth rate of the culture. Batch- and chemostat-cultivated cells from cultures with similar specific growth rates showed similar stress sensitivities. Generally, fast-growing cells were more sensitive to the stresses than slow-growing cells. For example, slow-growing chemostat-cultivated cells (D = 0.08 h−1) and stationary-phase bacteria from batch culture that were exposed to mild heat had very similar T90 (time until 90% of the population is inactivated) values (T90, chemostat = 2.66 h; T90, batch = 2.62 h), whereas T90 for cells growing at a μ of 0.9 h−1 was 0.2 h. We present evidence that the stress sensitivity of E. coli is correlated with the intracellular level of the alternative sigma factor RpoS. This is also supported by the fact that E. coli rpoS mutant cells were more stress sensitive than the parent strain by factors of 4.9 (mild heat), 5.3 (UVA light), and 4.1 (sunlight). Furthermore, modeling of inactivation curves with GInaFiT revealed that the shape of inactivation curves changed depending on the specific growth rate. Inactivation curves of cells from fast-growing cultures (μ = 1.0 h−1) that were irradiated with UVA light showed a tailing effect, while for slow-growing cultures (μ = 0.3 h−1), inactivation curves with shoulders were obtained. Our findings emphasize the need for accurate reporting of specific growth rates and detailed culture conditions in disinfection studies to allow comparison of data from different studies and laboratories and sound interpretation of the data obtained.

Measurement and interpretation of microbial adenosine tri-phosphate (ATP) in aquatic environments

There is a widespread need for cultivation-free methods to quantify viability of natural microbial communities in aquatic environments. Adenosine tri-phosphate (ATP) is the energy currency of all living cells, and therefore a useful indicator of viability. A luminescence-based ATP kit/protocol was optimised in order to detect ATP concentrations as low as 0.0001 nM with a standard deviation of <5%. Using this method, more than 100 water samples from a variety of aquatic environments (drinking water, groundwater, bottled water, river water, lake water and wastewater effluent) were analysed for extracellular ATP and microbial ATP in comparison with flow-cytometric (FCM) parameters. Microbial ATP concentrations ranged between 3% and 97% of total ATP concentrations, and correlated well (R(2)=0.8) with the concentrations of intact microbial cells (after staining with propidium iodide). From this correlation, we calculated an average ATP-per-cell value of 1.75x10(-10)nmol/cell. An even better correlation (R(2)=0.88) was observed between intact biovolume (derived from FCM scatter data) and microbial ATP concentrations, and an average ATP-per-biovolume value of 2.95x10(-9)nmol/microm(3) was calculated. These results support the use of ATP analysis for both routine monitoring and research purposes, and contribute towards a better interpretation of ATP data.

Overnight stagnation of drinking water in household taps induces microbial growth and changes in community composition.

Drinking water quality is routinely monitored in the distribution network but not inside households at the point of consumption. Fluctuating temperatures, residence times (stagnation), pipe materials and decreasing pipe diameters can promote bacterial growth in buildings. To test the influence of stagnation in households on the bacterial cell concentrations and composition, water was sampled from 10 separate households after overnight stagnation and after flushing the taps. Cell concentrations, measured by flow cytometry, increased (2-3-fold) in all water samples after stagnation. This increase was also observed in adenosine tri-phosphate (ATP) concentrations (2-18-fold) and heterotrophic plate counts (4-580-fold). An observed increase in cell biovolume and ATP-per-cell concentrations furthermore suggests that the increase in cell concentrations was due to microbial growth. After 5 min flushing of the taps, cell concentrations and water temperature decreased to the level generally found in the drinking water network. Denaturing gradient gel electrophoresis also showed a change in the microbial composition after stagnation. This study showed that water stagnation in household pipes results in considerable microbial changes. While hygienic risk was not directly assessed, it emphasizes the need for the development of good material validation methods, recommendations and spot tests for in-house water installations. However, a simple mitigation strategy would be a short flushing of taps prior to use.

Isolation and characterization of low nucleic acid (LNA)-content bacteria

Most planktonic bacteria are ‘uncultivable’ with conventional methods. Flow cytometry (FCM) is one approach that has been taken to study these bacteria. In natural aquatic environments, bacteria with high nucleic acid (HNA) and low nucleic acid (LNA) content are commonly observed with FCM after staining with fluorescent dyes. Although several studies have focused on the relative abundance and in situ activities of these two groups, knowledge on the growth of particularly LNA bacteria is largely limited. In this study, typical LNA bacteria were enriched from three different freshwater sources using extinction dilution (ED) and fluorescence-activated cell sorting (FACS). We have shown for the first time that LNA bacteria can be isolated and cultivated by using sterile freshwater as a growth medium. During growth, the typical LNA characteristics (that is, low-fluorescence intensity and sideward scatter (SSC)) remained distinct from those of typical HNA bacteria. Three LNA pure cultures that are closely affiliated to the Polynucleobacter cluster according to 16S rRNA sequencing results were isolated. Owing to their small size, cells of the isolates remained intact during cryo-transmission electronic microscopy examination and showed a Gram-negative cell-wall structure. The extremely small cell volume (0.05 μm3) observed for all three isolates indicates that they are among the smallest free-living heterotrophic organisms known in culture. Their isolation and cultivation allow further detailed investigation of this group of organisms under defined laboratory conditions.

Escherichia coli O157 can grow in natural freshwater at low carbon concentrations

Whereas much information on the die-off of Escherichia coli in the aquatic environment is available, only few data support its growth under such conditions. We therefore investigated batch growth in microcosms containing different types of sterile freshwater. The water samples were inoculated with low starting cell concentrations of E. coli O157 (3 x 10(3) cells ml(-1)) and growth was followed using nucleic acid staining combined with flow cytometry. We demonstrated that E. coli O157 is able to grow in sterile freshwater at low carbon concentrations, which is against the common view that cell numbers decline over time when added to freshwater samples. A correlation between apparent assimilable organic carbon (AOC(app)) concentration and the final cell concentration reached by E. coli O157 was established (P < 0.01). A considerable fraction of the AOC(app) (34 +/- 13%) was used by E. coli O157 but the numerical cell yield was about five-times lower in comparison with the bacterial AOC-test community, which originated from natural freshwater. On average, the maximum specific growth rate (mu(max)) of E. coli O157 growing in sterile freshwater at 30 degrees C was 0.19 +/- 0.07 h(-1). Batch growth assays at five different temperatures revealed a positive influence of temperature on mu(max) of E. coli O157. The results give new information on the behaviour of this common pathogen in the aquatic environment and contribute to microbial risk assessment in order to prevent spreading of water-borne diseases.