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Molecular evidence supporting the existence of two major groups in uropathogenic Escherichia coli.

Molecular methods allow an extremely fine strain typing that can be used to establish the population structure of bacterial species. This methodology has been used to characterize a collection of 74 uropathogenic Escherichia coli obtained from three hospitals located in geographically distant towns in Spain, some representatives of the ECOR collection and other reference strains. Genomic DNA was analyzed by RAPD (Random Amplified Polymorphic DNA) that can characterize a bacterial strain to the level of defining individual clones. The 16S rDNA-23S rDNA spacers were amplified by PCR and submitted to restriction analysis. Finally, the presence or absence of G adhesins in Escherichia coli as well as the type of adhesin (three types are known) have been shown by PCR amplification followed by digestion with restriction enzymes. As expected a wide diversity was shown by RAPD and identical patterns were only found in the case of strains isolated from the same individual, an obvious case of relapse. Analysis of the spacers' restriction patterns showed the presence of two markedly differentiated clusters that we have named alpha and beta. Both RAPD and spacer restriction patterns originated similar clusters of strains showing a consistency in the evolution of the global genome with the sequence variation of the ribosomal spacers. Furthermore, most of the strains having G-adhesin, with only a few exceptions, corresponded to the alpha rRNA spacer group. The two spacer types detected were also consistent with some phenotypic markers such as sucrose and raffinose utilization. The alpha and beta clusters could be intraspecific groups produced by partial sexual isolation or other barriers that are originating a divergent evolution.

Adhesins, Bacterial↗

Mechanism of action of EM 49, membrane-active peptide antibiotic.

EM 49 (recently renamed octapeptin) is a membrane-active peptide antibiotic that has been reported to affect the structure of bacterial membranes (K. S. Rosenthal, P. E. Swanson, and D. R. Storm, Biochemistry 15:5783-5792, 1976). In this study, it is shown that the effects of EM 49 on bacterial metabolism are similar to those of uncouplers of oxidative phosphorylation. EM 49 stimulated bacterial respiration within a narrow concentration range corresponding to minimum inhibitory concentrations and inhibited respiration at concentrations comparable to minimum biocidal concentrations. In addition, the peptide increased membrane proton permeability and lowered the adenosine 5'-triphosphate pool size. Parallel studies done with the related antibiotic polymyxin B demonstrated that the two peptides differed considerably in their effects on bacterial respiration. In contrast to EM 49, polymyxin B did not stimulate respiration at any concentration. It is proposed that the primary action of EM 49 is to disrupt the selective ion permeability of the cytoplasmic membrane, thereby relaxing the membrane potential.

Anti-Bacterial Agents↗

Improved fixation of cellulose-acetate reverse-osmosis membrane for scanning electron microscopy.

Fixation of cellulose-acetate membranes with either glutaraldehyde-osmium tetroxide or glutaraldehyde-ruthenium tetroxide resulted in extensive electron beam damage. Beam damage was eliminated and the bacterial surface structure was preserved, however, when cellulose-acetate membranes were fixed with glutaraldehyderuthenium tetroxide and treated successively with thiocarbohydrazide and osmium tetroxide.

Journal Article↗

Structure and dynamics of anaerobic bacterial aggregates in a gas-lift reactor.

Anaerobic mixed-culture aggregates, which converted glucose to acetic, propionic, butyric, and valeric acids, were formed under controlled conditions of substrate feed (carbon limitation) and hydraulic regimen. The continuous-flow system used (anaerobic gas-lift reactor) was designed to retain bacterial aggregates in a well-mixed reactor. Carrier availability (i.e., liquid-suspended sand grains) proved necessary for bacterial aggregate formation from individual cells during reactor start-up. Electron microscopic examination revealed that incipient colonization of sand grains by bacteria from the bulk liquid occurred in surface irregularities, conceivably reflecting local quiescence. Subsequent confluent biofilm formation on sand grains proved to be unstable, however. Substrate depletion in the bulk liquid is assumed to weaken deeper parts of the biofilm due to cellular lysis, after which production of gas bubbles and liquid shearing forces cause sloughing. The resulting fragments, although sand free, were nevertheless large enough to be retained in the reactor and gradually grew larger through bacterial growth and by clumping together with other fragments. In the final steady state, high cell densities were maintained in the form of aggregates, while sand had virtually disappeared due to sampling losses and wash-out. Numerical cell densities within aggregates ranged from 10/ml at the periphery to very low values in the center. The cells were enmeshed in a polymer matrix containing polysaccharides; nevertheless, carbon sufficiency was not a prerequisite to sustain high hold-up ratios.

Journal Article↗

Bacterial community dynamics during start-up of a trickle-bed bioreactor degrading aromatic compounds.

This study was performed with a laboratory-scale fixed-bed bioreactor degrading a mixture of aromatic compounds (Solvesso100). The starter culture for the bioreactor was prepared in a fermentor with a wastewater sample of a care painting facility as the inoculum and Solvesso100 as the sole carbon source. The bacterial community dynamics in the fermentor and the bioreactor were examined by a conventional isolation procedure and in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotides. Two significant shifts in the bacterial community structure could be demonstrated. The original inoculum from the wastewater of the car factory was rich in proteobacteria of the alpha and beta subclasses, while the final fermentor enrichment was dominated by bacteria closely related to Pseudomonas putida or Pseudomonas mendocina, which both belong to the gamma subclass of the class Proteobacteria. A second significant shift was observed when the fermentor culture was transferred as inoculum to the trickle-bed bioreactor. The community structure in the bioreactor gradually returned to a higher complexity, with the dominance of beta and alpha subclass proteobacteria, whereas the gamma subclass proteobacteria sharply declined. Obviously, the preceded pollutant adaptant did not lead to a significant enrichment of bacteria that finally dominated in the trickle-bed bioreactor. In the course of experiments, three new 16S as well as 23S rRNA-targeted probes for beta subclass proteobacteria were designed, probe SUBU1237 for the genera Burkholderia and Sutterella, probe ALBO34a for the genera Alcaligenes and Bordetella, and probe Bcv13b for Burkholderia cepacia and Burkholderia vietnamiensis. Bacteria hybridizing with the probe Bcv13b represented the main Solvesso100-degrading population in the reactor.

Bacteria↗

Effects of low temperature and freeze-thaw cycles on hydrocarbon biodegradation in Arctic tundra soil.

Degradation of petroleum hydrocarbons was monitored in microcosms with diesel fuel-contaminated Arctic tundra soil incubated for 48 days at low temperatures (-5, 0, and 7 degrees C). An additional treatment was incubation for alternating 24-h periods at 7 and -5 degrees C. Hydrocarbons were biodegraded at or above 0 degrees C, and freeze-thaw cycles may have actually stimulated hydrocarbon biodegradation. Total petroleum hydrocarbon (TPH) removal over 48 days in the 7, 0, and 7 and -5 degrees C treatments, respectively, was 450, 300, and 600 microg/g of soil. No TPH removal was observed at -5 degrees C. Total carbon dioxide production suggested that TPH removal was due to biological mineralization. Bacterial metabolic activity, indicated by RNA/DNA ratios, was higher in the middle of the experiment (day 21) than at the start, in agreement with measured hydrocarbon removal and carbon dioxide production activities. The total numbers of culturable heterotrophs and of hydrocarbon degraders did not change significantly over the 48 days of incubation in any of the treatments. At the end of the experiment, bacterial community structure, evaluated by ribosomal intergenic spacer length analysis, was very similar in all of the treatments but the alternating 7 and -5 degrees C treatment.

Arctic Regions↗

2-Bromoethanesulfonate affects bacteria in a trichloroethene-dechlorinating culture.

Long-term exposure to 2-bromoethanesulfonate (BES), an agent known to inhibit methanogenesis, altered the bacterial community structure of an anaerobic enrichment culture that reductively dechlorinated trichloroethene (TCE). BES did not hinder the dechlorination of TCE or other chlorinated ethenes as previously reported, although different intermediates and end products were observed.

Alkanesulfonic Acids↗

Relationship between bacterial community composition and bottom-up versus top-down variables in four eutrophic shallow lakes.

Bacterial community composition was monitored in four shallow eutrophic lakes during one year using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified prokaryotic rDNA genes. Of the four lakes investigated, two were of the clearwater type and had dense stands of submerged macrophytes while two others were of the turbid type characterized by the occurrence of phytoplankton blooms. One turbid and one clearwater lake had high nutrient levels (total phosphorus, >100 micro g liter(-1)) while the other lakes had relatively low nutrient levels (total phosphorus, <100 micro g liter(-1)). For each lake, seasonal changes in the bacterial community were related to bottom-up (resources) and top-down (grazers) variables by using canonical correspondence analysis (CCA). Using an artificial model dataset to which potential sources of error associated with the use of relative band intensities in DGGE analysis were added, we found that preferential amplification of certain rDNA genes over others does not obscure the relationship between bacterial community composition and explanatory variables. Besides, using this artificial dataset as well as our own data, we found a better correlation between bacterial community composition and explanatory variables by using relative band intensities compared to using presence/absence data. While bacterial community composition was related to phytoplankton biomass in the high-nutrient lakes no such relation was found in the low-nutrient lakes, where the bacterial community is probably dependent on other organic matter sources. We used variation partitioning to evaluate top-down regulation of bacterial community composition after bottom-up regulation has been accounted for. Using this approach, we found no evidence for top-down regulation of bacterial community composition in the turbid lakes, while grazing by ciliates and daphnids (Daphnia and Ceriodaphnia) was significantly related to changes in the bacterial community in the clearwater lakes. Our results suggest that in eutrophic shallow lakes, seasonality of bacterial community structure is dependent on the dominant substrate source as well as on the food web structure.

Animals↗

Activity and diversity of sulfate-reducing bacteria in a petroleum hydrocarbon-contaminated aquifer.

Microbial sulfate reduction is an important metabolic activity in petroleum hydrocarbon (PHC)-contaminated aquifers. We quantified carbon source-enhanced microbial SO(4)(2-) reduction in a PHC-contaminated aquifer by using single-well push-pull tests and related the consumption of sulfate and added carbon sources to the presence of certain genera of sulfate-reducing bacteria (SRB). We also used molecular methods to assess suspended SRB diversity. In four consecutive tests, we injected anoxic test solutions (1,000 liters) containing bromide as a conservative tracer, sulfate, and either propionate, butyrate, lactate, or acetate as reactants into an existing monitoring well. After an initial incubation period, 1,000 liters of test solution-groundwater mixture was extracted from the same well. Average total test duration was 71 h. We measured concentrations of bromide, sulfate, and carbon sources in native groundwater as well as in injection and extraction phase samples and characterized the SRB population by using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Enhanced sulfate reduction concomitant with carbon source degradation was observed in all tests. Computed first-order rate coefficients ranged from 0.19 to 0.32 day(-1) for sulfate reduction and from 0.13 to 0.60 day(-1) for carbon source degradation. Sulfur isotope fractionation in unconsumed sulfate indicated that sulfate reduction was microbially mediated. Enhancement of sulfate reduction due to carbon source additions in all tests and variability of rate coefficients suggested the presence of specific SRB genera and a high diversity of SRB. We confirmed this by using FISH and DGGE. A large fraction of suspended bacteria hybridized with SRB-targeting probes SRB385 plus SRB385-Db (11 to 24% of total cells). FISH results showed that the activity of these bacteria was enhanced by addition of sulfate and carbon sources during push-pull tests. However, DGGE profiles indicated that the bacterial community structure of the dominant species did not change during the tests. Thus, the combination of push-pull tests with molecular methods provided valuable insights into microbial processes, activities, and diversity in the sulfate-reducing zone of a PHC-contaminated aquifer.

Colony Count, Microbial↗

Empirical and theoretical bacterial diversity in four Arizona soils.

Understanding patterns of biodiversity in microbial communities is severely constrained by the difficulty of adequately sampling these complex systems. We illustrate the problem with empirical data from small surveys (200-member 16S rRNA gene clone libraries) of four bacterial soil communities from two locations in Arizona. Among the four surveys, nearly 500 species-level groups ( Dunbar et al., Appl. Environ. Microbiol. 65:662-1669, 1999) and 21 bacterial divisions were documented, including four new candidate divisions provisionally designated SC1, SC2, SC3, and SC4. We devised a simple approach to constructing theoretical null models of bacterial species abundance. These null models provide, for the first time, detailed descriptions of soil bacterial community structure that can be used to guide experimental design. Models based on a lognormal distribution were consistent with the observed sizes of the four communities and the richness of the clone surveys. Predictions from the models showed that the species richness of small surveys from complex communities is reproducible, whereas the species composition is not. By using the models, we can now estimate the required survey scale to document specified fractions of community diversity. For example, documentation of half the species in each model community would require surveys of 16,284 to 44,000 individuals. However, quantitative comparisons of half the species in two communities would require surveys at least 10-fold larger for each community.

Arizona↗

Effects of aeration cycles on nitrifying bacterial populations and nitrogen removal in intermittently aerated reactors.

The effects of the lengths of aeration and nonaeration periods on nitrogen removal and the nitrifying bacterial community structure were assessed in intermittently aerated (IA) reactors treating digested swine wastewater. Five IA reactors were operated in parallel with different aeration-to-nonaeration time ratios (ANA). Populations of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were monitored using 16S rRNA slot blot hybridizations. AOB species diversity was assessed using amoA gene denaturant gradient gel electrophoresis. Nitrosomonas and Nitrosococcus mobilis were the dominant AOB and Nitrospira spp. were the dominant NOB in all reactors, although Nitrosospira and Nitrobacter were also detected at lower levels. Reactors operated with the shortest aeration time (30 min) showed the highest Nitrosospira rRNA levels, and reactors operated with the longest anoxic periods (3 and 4 h) showed the lowest levels of Nitrobacter, compared to the other reactors. Nitrosomonas sp. strain Nm107 was detected in all reactors, regardless of the reactor's performance. Close relatives of Nitrosomonas europaea, Nitrosomonas sp. strain ENI-11, and Nitrosospira multiformis were occasionally detected in all reactors. Biomass fractions of AOB and effluent ammonia concentrations were not significantly different among the reactors. NOB were more sensitive than AOB to long nonaeration periods, as nitrite accumulation and lower total NOB rRNA levels were observed for an ANA of 1 h:4 h. The reactor with the longest nonaeration time of 4 h performed partial nitrification, followed by denitrification via nitrite, whereas the other reactors removed nitrogen through traditional nitrification and denitrification via nitrate. Superior ammonia removal efficiencies were not associated with levels of specific AOB species or with higher AOB species diversity.

Aerobiosis↗

Identification of cyanophage Ma-LBP and infection of the cyanobacterium Microcystis aeruginosa from an Australian subtropical lake by the virus.

Viruses can control the structure of bacterial communities in aquatic environments. The aim of this project was to determine if cyanophages (viruses specific to cyanobacteria) could exert a controlling influence on the abundance of the potentially toxic cyanobacterium Microcystis aeruginosa (host). M. aeruginosa was isolated, cultured, and characterized from a subtropical monomictic lake-Lake Baroon, Sunshine Coast, Queensland, Australia. The viral communities in the lake were separated from cyanobacterial grazers by filtration and chloroform washing. The natural lake viral cocktail was incubated with the M. aeruginosa host growing under optimal light and nutrient conditions. The specific growth rate of the host was 0.023 h(-1); generation time, 30.2 h. Within 6 days, the host abundance decreased by 95%. The density of the cyanophage was positively correlated with the rate of M. aeruginosa cell lysis (r(2) = 0.95). The cyanophage replication time was 11.2 h, with an average burst size of 28 viral particles per host cell. However, in 3 weeks, the cultured host community recovered, possibly because the host developed resistance (immunity) to the cyanophage. The multiplicity of infection was determined to be 2,890 virus-like particles/cultured host cell, using an undiluted lake viral population. Transmission electron microscopy showed that two types of virus were likely controlling the host cyanobacterial abundance. Both viruses displayed T7-like morphology and belonged to the Podoviridiae group (short tails) of viruses that we called cyanophage Ma-LBP. In Lake Baroon, the number of the cyanophage Ma-LBP was 5.6 x 10(4) cyanophage x ml(-1), representing 0.23% of the natural viral population of 2.46 x 10(7) x ml(-1). Our results showed that this cyanophage could be a major natural control mechanism of M. aeruginosa abundance in aquatic ecosystems like Lake Baroon. Future studies of potentially toxic cyanobacterial blooms need to consider factors that influence cyanophage attachment, infectivity, and lysis of their host alongside the physical and chemical parameters that drive cyanobacterial growth and production.

Australia↗

Adjuvant activity of synthetic N-acetylmuramyl peptides in rats.

The minimal adjuvant-active structure in bacterial cell wall peptidoglycan for the induction of delayed hypersensitivity in rats (WKA) was the N-acetylmuramyl dipeptides (N-acetylmuramyl L-alanyl-D-isoglutamine and N-acetylmuramyl L-alanyl-D-glutamic acid).

Adjuvants, Immunologic↗

Identification of Burkholderia cenocepacia genes required for bacterial survival in vivo.

Burkholderia cenocepacia (formerly Burkholderia cepacia complex genomovar III) causes chronic lung infections in patients with cystic fibrosis. In this work, we used a modified signature-tagged mutagenesis (STM) strategy for the isolation of B. cenocepacia mutants that cannot survive in vivo. Thirty-seven specialized plasposons, each carrying a unique oligonucleotide tag signature, were constructed and used to examine the survival of 2,627 B. cenocepacia transposon mutants, arranged in pools of 37 unique mutants, after a 10-day lung infection in rats by using the agar bead model. The recovered mutants were screened by real-time PCR, resulting in the identification of 260 mutants which presumably did not survive within the lungs. These mutants were repooled into smaller pools, and the infections were repeated. After a second screen, we isolated 102 mutants unable to survive in the rat model. The location of the transposon in each of these mutants was mapped within the B. cenocepacia chromosomes. We identified mutations in genes involved in cellular metabolism, global regulation, DNA replication and repair, and those encoding bacterial surface structures, including transmembrane proteins and cell surface polysaccharides. Also, we found 18 genes of unknown function, which are conserved in other bacteria. A subset of 12 representative mutants that were individually examined using the rat model in competition with the wild-type strain displayed reduced survival, confirming the predictive value of our STM screen. This study provides a blueprint to investigate at the molecular level the basis for survival and persistence of B. cenocepacia within the airways.

Animals↗

Organic solvents as probes for the structure and function of the bacterial membrane: effects of ethanol on the wild type and an ethanol-resistant mutant of Escherichia coli K-12.

The effects of ethanol on the growth of a wild-type Escherichia coli K-12 are described. These effects include a reduction of the steady-state growth rate and an interference with the division process. They appear as an immediate response to the addition of ethanol and are rapidly reversed by removal of ethanol. Mutants were selected that could grow at a concentration of ethanol that stopped wild-type growth. The growth of one of the mutants we studied (strain S9L100) is stimulated by the presence of ethanol, methanol, or dimethyl sulfoxide. This strain exhibits pleiotropic growth defects including abnormal cell division and morphology. It also appears to have an altered lac permease function which is not due to a mutation in the Y gene itself. We conclude that this mutant has an altered membrane and that the membrane defect may be the cause of the abnormal growth properties. The use of compounds which serve as general membrane perturbants and mutants resistant to these perturbants form a system accessible to both genetic and physical-biochemical techniques.

Bacterial Proteins↗

Morphological and cell association characteristics of Rochalimaea quintana: comparison of the Vole and Fuller strains.

The vole and Fuller strains of Rochalimaea quintana were grown on monolayers of mouse L cells irradiated 7 days previously and examined by light microscopy and scanning and transmission electron microscopy. Most of the bacteria of both strains were shown to adhere to the L cells but remained in an extracellular location. Cell division was frequently seen among the extracellular bacteria. The few intracellular bacteria seemed to be within vacuoles and did not multiply. Attachment to the eucaryotic cell did not seem to involve pili or other bacterial surface structures. The dimensions of the bacteria were approximately 0.45 micron in width by 1.0 to 1.7 micron in length. The cell envelope consisted of the usual trilaminar cell wall and plasma membranes separated by a layer of low electron density, as found in other gram-negative bacteria. No significant differences between the vole and Fuller strains either in morphology or relationship to eucaryotic cells were encountered.

Cell Membrane↗

Effects of the membrane action of tetralin on the functional and structural properties of artificial and bacterial membranes.

Tetralin is toxic to bacterial cells at concentrations below 100 mumol/liter. To assess the inhibitory action of tetralin on bacterial membranes, a membrane model system, consisting of proteoliposomes in which beef heart cytochrome c oxidase was reconstituted as the proton motive force-generating mechanism, and several gram-positive and gram-negative bacteria were studied. Because of its hydrophobicity, tetralin partitioned into lipid membranes preferentially (lipid/buffer partition coefficient of tetralin is approximately 1,100). The excessive accumulation of tetralin caused expansion of the membrane and impairment of different membrane functions. Studies with proteoliposomes and intact cells indicated that tetralin makes the membrane permeable for ions (protons) and inhibits the respiratory enzymes, which leads to a partial dissipation of the pH gradient and electrical potential. The effect of tetralin on the components of the proton motive force as well as disruption of protein-lipid interaction(s) could lead to impairment of various metabolic functions and to low growth rates. The data offer an explanation for the difficulty in isolating and cultivating microorganisms in media containing tetralin or other lipophilic compounds.

Arthrobacter↗