The Mechanism of Fractal-Like Structure Formation by Bacterial Populations

Three types of population growth and development of chemotaxic motile bacteria Escherichia coli on semi-solid nutrient media are investigated: a) stable development – circular symmetrical waves; b) bursts; c) fractal-like self-organization. Experimental investigation of the burst formation is presented. The microscopic analysis of growing, fractal-like structures is carried out, and a mechanism for such structure formation is suggested. It is supposed that fractal-like bacterial structures growth is based on the principle of successively forming multiple micro-bursts. A mathematical model has been suggested to reproduce the experimental results. The structures obtained by numerical modeling of population growth in the parameter space substrate concentration - bacterial movement rate reproduce the corresponding experimental structures in the space nutrient concentration in the media – the density of the media.     

The Response of Three Bacterial Populations to Pollution in a Stream

Abstract Practical methods for biomonitoring of natural systems are still under development. Bacteria are potentially useful indicators of water quality because of their species diversity and ability to rapidly respond to changing environmental conditions. In this study, bacterial populations from unpolluted and polluted stream sites in two watersheds were compared to determine their suitability for use as environmental indicators. Upper Three Runs Creek and Four Mile Creek headwaters have had little anthropogenic disturbance, as opposed to lower Four Mile Creek which received thermal, radioactive, and chemical perturbations. Chemical and physical measurements provided evidence that seepage from holding ponds polluted Four Mile Creek. Polluted sites did not have altered total bacterial numbers but had decreased numbers of colony-forming units. Abundances of three bacterial species, Acinetobacter calcoaceticus, Burkholderia cepacia, and Pseudomonas putida, were determined by colony hybridization with species-specific rDNA probes. Contribution of A. calcoaceticus to the assemblage was higher at polluted sites, which indicated either tolerance of polluted conditions or the ability to utilize compounds existing at these sites to reach larger populations. No differences in B. cepacia populations were detected, and differences in P. putida populations could not be attributed solely to disturbance. The pollution of Four Mile Creek induced differences in bacterial populations that could be monitored using the described approach. Received: 24 September 1996; Accepted: 20 December 1996     

Abundance of Three Bacterial Populations in Selected Streams

The population sizes of three bacterial species, Acinetobacter calcoaceticus, Burkholderia cepacia, and Pseudomonas putida, were examined in water and sediment from nine streams in different parts of the United States using fluorescent in situ hybridization (FISH). Population sizes were determined from three sites (upstream, midstream, and downstream) in each stream to compare differences in the occurrence and distribution of the species within each stream and among streams. Physical and chemical variables measured reflected differences in environmental conditions among the streams. In the water, B. cepacia numbers were highest in the agricultural, Iowa stream. P. putida numbers were highest in the southern coastal plain streams, Black Creek (GA) and Meyers Branch (SC). Compared to the other two species, the abundance of A. calcoaceticus was similar in all the streams. In sediment, the greatest abundance of all three species was found in the Iowa stream, while the lowest was in Hugh White Creek (NC). Detrended correspondence analysis (DCA) explained 95.8% and 83.9% of the total variation in bacterial numbers in water and sediment of the streams, respectively. In sediments and water, B. cepacia numbers were related to nitrate concentrations. A. calcoaceticus in water clustered with several environmental variables (i.e., SRP, pH, and conductivity) but benthic populations were less well correlated with these variables. This study reveals the potential influence of various environmental conditions on different bacterial populations in stream communities.     

Bacterial Diversity in a Nonsaline Alkaline Environment: Heterotrophic Aerobic Populations

Heterotrophic populations were isolated and characterized from an alkaline groundwater environment generated by active serpentinization, which results in a Ca(OH)₂-enriched, extremely diluted groundwater with pH 11.4. One hundred eighty-five strains were isolated in different media at different pH values during two sampling periods. To assess the degree of diversity present in the environment and to select representative strains for further characterization of the populations, we screened the isolates by using random amplified polymorphic DNA-PCR profiles and grouped them based on similarities determined by fatty acid methyl ester analysis. Phenotypic characterization, determinations of G+C content, phylogenetic analyses by direct sequencing of 16S rRNA genes, and determinations of pH tolerance were performed with the selected isolates. Although 38 different populations were identified and characterized, the vast majority of the isolates were gram positive with high G+C contents and were affiliated with three distinct groups, namely, strains closely related to the species Dietzia natrolimnae (32% of the isolates), to Frigoribacterium/Clavibacter lineages (29% of the isolates), and to the type strain of Microbacterium kitamiense (20% of the isolates). Other isolates were phylogenetically related to strains of the genera Agrococcus, Leifsonia, Kytococcus, Janibacter, Kocuria, Rothia, Nesterenkonia, Citrococcus, Micrococcus, Actinomyces, Rhodococcus, Bacillus, and Staphylococcus. Only five isolates were gram negative: one was related to the Sphingobacteria lineage and the other four were related to the α-Proteobacteria lineage. Despite the pH of the environment, the vast majority of the populations were alkali tolerant, and only two strains were able to grow at pH 11.     

Defining TCE Plume Source Areas Using the Membrane Interface Probe (MIP)

A variety of traditional characterization methods, such as soil gas surveys, soil and groundwater sampling, and fixed laboratory analysis, is commonly used to define the magnitude and extent of soil and groundwater contamination in volatile organic compound source areas. One significant limitation of these methods is that they require multi-media sample collection to define the full unsaturated and saturated vertical profile in any given location. In addition, attaining higher resolution by increasing sample frequency increases costs substantially. A relatively new technology, the Membrane Interface Probe (MIP), was used to define trichloroethene plume source areas at F.E. Warren Air Force Base in Cheyenne, Wyoming, and at a confidential security products manufacturer in Tennessee. The MIP offered significant advantages over traditional drilling and direct-push methods and yielded data critical to a fuller understanding of subsurface conditions. The near-continuous MIP profile minimized the number of soil and groundwater samples required to fully delineate the extent of the plume-head source areas. In addition, the MIP is also able to collect data in the vadose and saturated zones, providing detailed vertical contaminant profiling information and geologic conditions based on soil conductivity that aided in the development of the site conceptual model. The MIP was not without its disadvantages; principal among these the relatively high detection limit (approximately 100 ppb in soil and groundwater), making the method useful for source characterization but limited for delineating lower levels of contamination. The data obtained from the MIP are considered screening-level data and need to be supplemented with analytical soil or groundwater data to fully support risk or remediation decisions. In summary, the vertical profiling obtained using the MIP aided in the interpretation of the complex relationship between the presence of gross contamination in soil and groundwater and the geologic conditions controlling contaminant distribution.     

Overview of Mathematical Approaches Used to Model Bacterial Chemotaxis II: Bacterial Populations

We review the application of mathematical modeling to understanding the behavior of populations of chemotactic bacteria. The application of continuum mathematical models, in particular generalized Keller–Segel models, is discussed along with attempts to incorporate the microscale (individual) behavior on the macroscale, modeling the interaction between different species of bacteria, the interaction of bacteria with their environment, and methods used to obtain experimentally verified parameter values. We allude briefly to the role of modeling pattern formation in understanding collective behavior within bacterial populations. Various aspects of each model are discussed and areas for possible future research are postulated.     

Active Bacterial Populations and Grazing Impact Revealed by an In Situ Experiment in a Shallow Aquifer

To elucidate bacterial population dynamics in an aquifer, we attempted to reveal the impact of protozoan grazing on bacterial productivity and community structure by an in situ incubation experiment using a diffusion chamber. The abundance and vertical distribution of bacteria and protozoa in the aquifer were revealed using wells that were drilled in a sedimentary rock system in Itako, Ibaraki, Japan. The water column in the wells possessed aerobic and anaerobic layers. Active bacterial populations under the grazing pressure of protozoa were revealed through in situ incubation with grazer eliminating experiment by the filtration. On August 19, 2003, the total number of bacteria (TDC) decreased from 1.5 × 10⁶ cells ml^{? 1} at 2.2 m depth to 3.0 × 10⁵ cells ml^{? 1} at 10 m depth. The relative contribution of the domain Bacteria to TDC ranged between 63% and 84%. Protozoa existed at a density of 4.2 × 10⁴ to 1.9 × 10⁵ cells ml^{? 1} in both aerobic and microaerobic conditions. A grazing elimination experiment in situ for 6 days brought about clearly different bacterial community profiles between the 2.2 m and 10 m samples. The bacterial composition of the initial community was predominantly β- and γ -proteobacteria at 2.2 m, while at 10 m β-, α - and γ -proteobacteria represented 56%, 26% and 13% of the community, respectively. The distribution of bacterial abundance, community composition and growth rates in the subsurface were influenced by grazing as well as by geochemical factors (dissolved oxygen and concentrations of organic carbon, methane and sulfate). Results of the in situ incubation experiment suggested that protozoan grazing contributes significantly to bacterial population dynamics.     

Environmental Factors Correlated with Size of Bacterial Populations in a Polluted Stream

Samples of water were taken from a polluted zone of the Gallinas River and analyzed as to numbers of total bacteria, coliforms, and fecal streptococci. Environmental factors measured were temperature, pH and concentrations of detergent, nitrate plus nitrite nitrogen, sulfate, chloride, bicarbonate, and phosphate. Thirty-two observations were made from 12 March through 22 July 1971. Stepwise multiple linear regression analyses of the data were carried out by computer to determine which of the environmental factors were significantly correlated with numbers of bacteria present. A multiple linear regression equation was constructed for each bacteriological parameter as a function of significant variables only. Log total bacteria was correlated positively with bicarbonate, phosphate, and detergent concentrations. Log coliforms was correlated positively with phosphate and sulfate concentrations and negatively with chloride concentration. Log fecal streptococci was correlated positively with bicarbonate and chloride concentrations.     

Distribution and Life Strategies of Two Bacterial Populations in a Eutrophic Lake

Monoclonal antibodies and epifluorescence microscopy were used to determine the depth distribution of two indigenous bacterial populations in the stratified Lake Plußsee and characterize their life strategies. Populations of Comamonas acidovorans PX54 showed a depth distribution with maximum abundances in the oxic epilimnion, whereas Aeromonas hydrophila PU7718 showed a depth distribution with maximum abundances in the anoxic thermocline layer (metalimnion), i.e., in the water layer with the highest microbial activity. Resistance of PX54 to protist grazing and high metabolic versatility and growth rate of PU7718 were the most important life strategy traits for explaining the depth distribution of the two bacterial populations. Maximum abundance of PX54 was 16,000 cells per ml, and maximum abundance of PU7718 was 20,000 cells per ml. Determination of bacterial productivity in dilution cultures with different-size fractions of dissolved organic matter (DOM) from lake water indicates that low-molecular-weight (LMW) DOM is less bioreactive than total DOM (TDOM). The abundance and growth rate of PU7718 were highest in the TDOM fractions, whereas those of PX54 were highest in the LMW DOM fraction, demonstrating that PX54 can grow well on the less bioreactive DOM fraction. We estimated that 13 to 24% of the entire bacterial community and 14% of PU7718 were removed by viral lysis, whereas no significant effect of viral lysis on PX54 could be detected. Growth rates of PX54 (0.11 to 0.13 h⁻¹) were higher than those of the entire bacterial community (0.04 to 0.08 h⁻¹) but lower than those of PU7718 (0.26 to 0.31 h⁻¹). In undiluted cultures, the growth rates were significantly lower, pointing to density effects such as resource limitation or antibiosis, and the effects were stronger for PU7718 and the entire bacterial community than for PX54. Life strategy characterizations based on data from literature and this study revealed that the fast-growing and metabolically versatile A. hydrophila PU7718 is an r-strategist or opportunistic population in Lake Plußsee, whereas the grazing-resistant C. acidovorans PX54 is rather a K-strategist or equilibrium population.     

Deposition Gradients Near to a Point Source in a Barley Crop

By measuring deposits of droplets downwind from a source in a barley crop during crop growth, gradients of deposition were established. Droplets were generated using a May spinning disc, which approximated to a point source. Droplets were labelled with thiabendazole so that deposit could be measured photometrically. Droplet diameter was approximately 20 micrometres, a size similar to spores of barley powdery mildew, an important foliar pathogen. Gradients of deposition were influenced by the density of the crop, by wind speed and by air turbulence. At any one time these three factors could interact to change the gradient substantially. Exponential and power law equations fitted the data equally well although due to experimental variation neither gave a very good fit. It is suggested that over the first few metres of dispersal from the source an exponential equation can be used to model the gradient. This has the advantage that it can be extrapolated to give an estimate of “self-infection” i.e. the spores deposited on the plant on which they were produced.     

Bacterial Populations Associated with Different Regions of the Human Colon Wall

The microorganisms associated with the undiseased human colon wall were examined in material obtained from four sudden-death victims. In traffic accident subjects (aged 45 and 16 years) the anaerobe-aerobe ratio was about 10⁴:1 in all areas of the colon examined, whereas in acute heart failure subjects (aged 74 and 46 years) the ratio was as low as 1.2:1. The flora of each individual was distinct and complex. Although the predominant anaerobes isolated were Bacteroides and Fusobacterium spp., which composed over 50% of the flora in some samples, the species isolated (indicated by morphology and glucose fermentation products) varied between individuals. Other major types observed were gram-positive nonsporing rods, including Bifidobacterium spp., and anaerobic cocci (between 8 and 20% of isolates). Clostridia were only isolated in significant numbers from one individual. Scanning electron microscopy showed that most of the organisms were present below the top surface of the mucin layer overlying the mucosa. The use of several different preparative procedures for microscopy showed a complex microbial structure within the mucus, but major variations in the bacterial populations in different areas of the colon were not found. Spiral-shaped organisms up to 60 μm long in the form of double helices were found in two subjects by scanning electron microscopy but were not isolated during the parallel bacteriological investigation. The differences between this and previous studies are discussed in relation to experimental procedures and also in contrast to results with animals that showed a particularly specialized flora associated with the colon wall.     

Soil Drying As a Model for the Action of Stress Factors on Natural Bacterial Populations

The drying of soil samples reduced the abundance (especially of predominant species) and the diversity of bacteria isolated from these samples, making easier the isolation of rare bacterial species. Some bacterial species that were minor before soil drying became dominant in dried soil samples. In general, soil drying allowed the diversity of soil bacteria to be determined more adequately. The bacteria that were isolated from dried soil samples turned out to be resistant to gamma radiation (with LD₉₀ = 2.8–4.6 kGy) and desiccation. It is concluded that soil drying may serve as a model for the action of stress factors on natural bacterial populations. The hypothesis that periodic desiccation was the primary cause of formation of bacterial radioresistance in nature is discussed.     

Plants as a Source of Bacterial Resistance Modulators and Anti-Infective Agents

The spread of multidrug-resistant (MDR) strains of bacteria necessitates the discovery of new classes of antibacterials and compounds that inhibit these resistance mechanisms. At present, there are no single chemical entity plant-derived antibacterials used clinically, and this chemically diverse group deserves consideration as a source for two major reasons. First, plants have exceptional ability to produce cytotoxic agents and second there is an ecological rationale that antimicrobial natural products should be present or synthesised de novo in plants following microbial attack to protect the producer from pathogenic microbes in its environment. We have been characterising plant-derived products that are either antibacterial in their own right, or modulators of resistance in bacterial strains possessing multidrug efflux mechanisms. These efflux transporters are responsible for resistance to certain antibiotics and antiseptics and occur in strains of methicillin-resistant Staphylococcus aureus (MRSA), a major clinical problem at present. We are also investigating plant sources for compounds with activity against mycobacteria with a view to discovering drug leads with potential activity toward tuberculosis (TB) producing species. This paper will briefly review the literature on plant derived bacterial resistance modifying agents and antibacterials. Examples in this area from our own work will be given. The activities of plant-derived antibacterials show that there are many potential new classes of antibacterial agents which should undergo further cytotoxicity, microbial specificity and preclinical studies.     

Distribution and Biogeochemical Importance of Bacterial Populations in a Thick Clay-Rich Aquitard System

To investigate the distribution of microbial biomass, populations and activities within a clay-rich, low hydraulic conductivity (10⁻¹¹ to 10⁻¹² m s⁻¹) aquitard complex, cores were aseptically obtained from a series of overlying clayey deposits; a fractured till, unfractured till (20–30 ka BP), a disturbed interfacial zone (20–30 ka BP), and a Cretaceous clay aquitard (71–72 Ma BP). The results of confocal microscopy studies, culture methods, molecular approaches, and extractive fatty acid analyses all indicated low bacterial numbers that were non-homogeneously distributed within the sediments. Various primers for catabolic genes were used to amplify extracted DNA. Results indicated the presence of eubacterial 23S rDNA, and thenarH gene for nitrate reductase and ribulose-1,5-biphosphate carboxylase (RuBP carboxylase). Although there was no evidence of limitation by electron acceptors or donors, sulfate-reducing bacteria were not detected below the fractured till zone, using PCR, enrichment, or culture techniques. Denaturing gradient gel electrophoresis (DGGE) analyses indicated differences in community composition and abundance between the various geologic units. Results of FAME analyses of sediments yielded detectable extractable fatty acids throughout the aquitard complex. Bacterial activities were demonstrated by measuring mineralization of (¹⁴C) glucose. Porewater chemistry and stable isotope data were in keeping with an environment in which extremely slow growing, low populations of bacteria exert little impact. The observations also support the contention that in low permeability sediments bacteria may survive for geologic time periods.     

Lognormal Distribution of Epiphytic Bacterial Populations on Leaf Surfaces

Total populations of epiphytic bacteria and selected components thereof were determined on sets of 24 to 36 individual leaves (corn, rye) or leaflets (snap bean, soybean, tomato) of field-grown plants by washing and dilution plating. In general, levels of component populations (e.g., bacteria that are ice nucleation active) were quantitatively more variable from leaf to leaf within a set than were total epiphytic bacterial populations. Populations of a given component frequently varied by a factor of 100 to 1,000 within a set of leaves. Total bacterial populations usually varied by a factor of about 10. For each set of leaves, total and component epiphytic bacterial populations were found to approximate a lognormal distribution by the Shapiro-Wilk test for normality. Due to the lognormal distribution of epiphytic bacterial populations, estimates of population size based on the common practice of using bulked samples (wherein several leaves are washed together) will overestimate the population median by a factor of approximately 1.15σ². From the known probability distribution of bacterial populations, the frequency with which a given bacterial population size is met or exceeded on individual leaves can be estimated. If the bacterial component is phytopathogenic, the frequency estimates could be used to relate quantitatively pathogen populations and disease incidence.