Transport of bacteria in porous media and its enhancement by surfactants for bioaugmentation: A review

The success of bioaugmentation processes for groundwater bioremediation requires efficient transport of bacteria in the subsurface environment. In this paper, the factors that influence transport of bacterial cells in porous media are reviewed and the effects of surfactants on the transport are discussed. Movement of bacterial cells in porous media is a process driven by advection and hydrodynamic dispersion forces of fluids. Immobilization of bacterial cells takes place due to processes such as adsorption and straining. Blocking and ripening along with bacterial migration process decrease and increase the retention of cells in porous media, respectively. Physicochemical properties of the porous media, groundwater chemistry, and properties of the bacterial cells affect the transport behavior. Surfactants have the potential to modify bacterial surface properties for both bacterial cells and medium solids, and thus enhance bacterial transport.     

Can dead bacterial cells be defined and are genes expressed after cell death?

There is a paucity of knowledge on gene expression in dead bacterial cells. Why would this knowledge be useful? The cells are dead. However, the time duration of gene expression following cell death is often unknown, and possibly in the order of minutes. In addition, it is a challenge to determine if bacterial cells are dead, or viable but non-culturable (VBNC), and what is an agreed upon correct definition of dead bacteria. Cells in the bacterial population or community may die at different rates or times and this complicates both the viability and gene expression analysis. In this article, the definition of dead bacterial cells is discussed and its significance in continued gene expression in cells following death. The definition of living and dead has implications for possible, completely, synthetic bacterial cells that may be capable of growth and division.     

A new method to determine initial viability of entrapped cells using fluorescent nucleic acid staining

Entrapped bacterial cells are widely used in several biotechnological applications. Cell entrapment procedures are known to affect the viability of bacterial cells. To determine the effect of entrapment procedures on viability of bacterial cells, dissolution of the entrapment matrices using chelating agents or heat is required immediately after the entrapment is completed. Chelating agents and heat applied in the matrix dissolution reduce cell viability and in turn hinder accurate quantification of viable cells. In this study, a method to determine the effect of entrapment procedure on bacterial cell viability which involves entrapping cells directly onto glass slides was developed. The developed method showed less viability reduction than the methods requiring matrix dissolution. The percentage of live cells in the culture before entrapment ranged from 54% to 74%, while the percent of live cells after entrapment determined by the developed method was 39-62%.     

Pore-forming toxins and cellular non-immune defenses (CNIDs)

Pore-forming toxins (PFTs) are the most common class of bacterial protein toxin and are important for bacterial pathogenesis. Recent studies have shown that the previous model stating that epithelial cells lyse in response to these toxins and have no defenses against these pores is oversimplified. Rather, it appears that cells have sophisticated mechanisms and signal-transduction pathways with which to respond to such an attack. There is a growing body of knowledge about how cells respond to and protect themselves against PFTs; this protection against PFTs is likely to be important in host survival to attack by bacterial pathogens, but does not neatly fit into current concepts of adaptive or innate immunity. Therefore, it is proposed that the terminology cellular non-immune defenses (CNIDs) be used to describe defenses that are employed by non-immune cells to protect against bacterial attack.     

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.     

Species-sorting may explain an apparent minimal effect of immigration on freshwater bacterial community dynamics

Long distance atmospheric transport of bacterial cells is often implied as a driver of the apparent cosmopolitan distribution of bacterial taxa. Surprisingly, efforts to measure immigration in bacterial communities are rare. An 8-week time series of within-lake bacterial community composition and atmospheric deposition rates and composition were used to estimate the influence of immigration on bacterial community dynamics in two north temperate lakes. Characterization of bacterial community dynamics using automated ribosomal intergenic spacer analysis suggested moderate overlap in composition between the lakes and atmospherically deposited cells. However, taxa that appeared to be delivered by atmospheric deposition had a relatively minor influence on lake bacterial community dynamics. The weak influence of immigrating bacterial taxa suggests that a species-sorting concept best describes aquatic bacterial metacommunity dynamics.     

Eukaryotic and prokaryotic signal peptides direct secretion of a bacterial endoglucanase by mammalian cells

It is well established that hydrophobic signal sequences direct proteins into or across the endoplasmic reticulum membrane in eukaryotes and cell membranes in prokaryotes. Although it is recognized that eukaryote proteins are efficiently secreted by bacterial systems, the export of bacterial proteins by eukaryotes has received little attention. To investigate membrane translocation of bacterial proteins by mammalian cells, the secretion of a bacterial endoglucanase (endoglucanase E) from stably transfected Chinese hamster ovary cells has been examined. We report that a functional endoglucanase is secreted when fused to prokaryote or eukaryote signal peptides. Furthermore, the endoglucanase was post-translationally modified before secretion. Data presented in this paper suggest that secretion of bacterial proteins by eukaryote cells may be a general phenomenon and infer that there are no specific requirements with respect to the origin of the signal sequences.     

Resolving chromosome segregation in bacteria

Bacterial chromosomes are evenly distributed between daughter cells, however no equivalent eukaryotic mitotic apparatus has been identified yet. Nevertheless, an advance in our understanding of the dynamics of the bacterial chromosome has been accomplished in recent years by adopting fluorescence microscopy techniques to visualize living bacterial cells. Here, some of the most recent studies that yield new insights into the nature of bacterial chromosome dynamics are described. In addition, we review in detail the current models that attempt to illuminate the mechanism of chromosome segregation in bacteria and discuss the possibility that a bacterial mitotic apparatus does indeed exist.     

Respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species- and cell type-dependent manner

Secondary bacterial infections often complicate respiratory viral infections, but the mechanisms whereby viruses predispose to bacterial disease are not completely understood. We determined the effects of infection with respiratory syncytial virus (RSV), human parainfluenza virus 3 (HPIV-3), and influenza virus on the abilities of nontypeable Haemophilus influenzae and Streptococcus pneumoniae to adhere to respiratory epithelial cells and how these viruses alter the expression of known receptors for these bacteria. All viruses enhanced bacterial adhesion to primary and immortalized cell lines. RSV and HPIV-3 infection increased the expression of several known receptors for pathogenic bacteria by primary bronchial epithelial cells and A549 cells but not by primary small airway epithelial cells. Influenza virus infection did not alter receptor expression. Paramyxoviruses augmented bacterial adherence to primary bronchial epithelial cells and immortalized cell lines by up-regulating eukaryotic cell receptors for these pathogens, whereas this mechanism was less significant in primary small airway epithelial cells and in influenza virus infections. Respiratory viruses promote bacterial adhesion to respiratory epithelial cells, a process that may increase bacterial colonization and contribute to disease. These studies highlight the distinct responses of different cell types to viral infection and the need to consider this variation when interpreting studies of the interactions between respiratory cells and viral pathogens.     

A small molecule that disrupts S. Typhimurium membrane voltage without cell lysis reduces bacterial colonization of mice

As pathogenic bacteria become increasingly resistant to antibiotics, antimicrobials with mechanisms of action distinct from current clinical antibiotics are needed. Gram-negative bacteria pose a particular problem because they defend themselves against chemicals with a minimally permeable outer membrane and with efflux pumps. During infection, innate immune defense molecules increase bacterial vulnerability to chemicals by permeabilizing the outer membrane and occupying efflux pumps. Therefore, screens for compounds that reduce bacterial colonization of mammalian cells have the potential to reveal unexplored therapeutic avenues. Here we describe a new small molecule, D66, that prevents the survival of a human Gram-negative pathogen in macrophages. D66 inhibits bacterial growth under conditions wherein the bacterial outer membrane or efflux pumps are compromised, but not in standard microbiological media. The compound disrupts voltage across the bacterial inner membrane at concentrations that do not permeabilize the inner membrane or lyse cells. Selection for bacterial clones resistant to D66 activity suggested that outer membrane integrity and efflux are the two major bacterial defense mechanisms against this compound. Treatment of mammalian cells with D66 does not permeabilize the mammalian cell membrane but does cause stress, as revealed by hyperpolarization of mitochondrial membranes. Nevertheless, the compound is tolerated in mice and reduces bacterial tissue load. These data suggest that the inner membrane could be a viable target for anti-Gram-negative antimicrobials, and that disruption of bacterial membrane voltage without lysis is sufficient to enable clearance from the host.     

Laser microdissection: exploring host-bacterial encounters at the front lines

The mucosal surfaces of tissues such as the stomach and intestines are in constant contact with indigenous bacterial populations and are major portals of entry for bacterial pathogens. Host responses to bacterial encounters at these surfaces frequently involve complex interactions between epithelial cells and immune cells, and are thus difficult to model in vitro. Laser microdissection is a technique in which pure populations of host cells are acquired from sections of complex tissue. When coupled with an expanding repertoire of techniques for molecular analysis of microdissected cells, laser microdissection allows host cellular responses to bacteria to be studied in their native tissue context. This approach has already yielded key insights into the nature of mucosal responses to commensal, as well as pathogenic bacteria, and promises to be an important addition to the cellular microbiologist's toolkit.     

Gamma delta-T cells are critical for survival and early proinflammatory cytokine gene expression during murine Klebsiella pneumonia

Although cells of the innate inflammatory response, such as macrophages and neutrophils, have been extensively studied in the arena of Gram-negative bacterial pneumonia, a role for T cells remains unknown. To study the role of specific T cell populations in bacterial pneumonia, mice deleted of their TCR beta- and/or delta-chain were intratracheally inoculated with Klebsiella pneumoniae. Gamma delta T cell knockout mice displayed increased mortality at both early and late time points. In contrast, mice specifically lacking only alpha beta-T cells were no more susceptible than wild-type mice. Pulmonary bacterial clearance in gamma delta-T cell knockout mice was unimpaired. Interestingly, these mice displayed increased peripheral blood dissemination. Rapid up-regulation of IFN-gamma and TNF-alpha gene expression, critical during bacterial infections, was markedly impaired in lung and liver tissue from gamma delta-T cell-deficient mice 24 h postinfection. The increased peripheral blood bacterial dissemination correlated with impaired hepatic bacterial clearance following pulmonary infection and increased hepatic injury as measured by plasma aspartate aminotransferase activity. Combined, these data suggest that mice lacking gamma delta-T cells have an impaired ability to resolve disseminated bacterial infections subsequent to the initial pulmonary infection. These data indicate that gamma delta-T cells comprise a critical component of the acute inflammatory response toward extracellular Gram-negative bacterial infections and are vital for the early production of the proinflammatory cytokines IFN-gamma and TNF-alpha.     

Large fraction of dead and inactive bacteria in coastal marine sediments: comparison of protocols for determination and ecological significance

It is now universally recognized that only a portion of aquatic bacteria is actively growing, but quantitative information on the fraction of living versus dormant or dead bacteria in marine sediments is completely lacking. We compared different protocols for the determination of the dead, dormant, and active bacterial fractions in two different marine sediments and at different depths into the sediment core. Bacterial counts ranged between (1.5 +/- 0.2) x 10(8) cells g(-1) and (53.1 +/- 16.0) x 10(8) cells g(-1) in sandy and muddy sediments, respectively. Bacteria displaying intact membrane (live bacterial cells) accounted for 26 to 30% of total bacterial counts, while dead cells represented the most abundant fraction (70 to 74%). Among living bacterial cells, nucleoid-containing cells represented only 4% of total bacterial counts, indicating that only a very limited fraction of bacterial assemblage was actively growing. Nucleoid-containing cells increased with increasing sediment organic content. The number of bacteria responsive to antibiotic treatment (direct viable count; range, 0.3 to 4.8% of the total bacterial number) was significantly lower than nucleoid-containing cell counts. An experiment of nutrient enrichment to stimulate a response of the dormant bacterial fraction determined a significant increase of nucleoid-containing cells. After nutrient enrichment, a large fraction of dormant bacteria (6 to 11% of the total bacterial number) was "reactivated." Bacterial turnover rates estimated ranged from 0.01 to 0.1 day(-1) but were 50 to 80 times higher when only the fraction of active bacteria was considered (on average 3.2 day(-1)). Our results suggest that the fraction of active bacteria in marine sediments is controlled by nutrient supply and availability and that their turnover rates are at least 1 order of magnitude higher than previously reported.     

Comparative proteomics study reveals that bacterial CpG motifs induce tumor cell autophagy in vitro and in vivo

Unmethylated CpG dinucleotides, present in bacterial DNA, are recognized in vertebrates via the Toll-like receptor 9 (TLR9) and are known to act as an anticancer agent by stimulating immune cells to induce a proinflammatory response. Although the effects of CpG-oligodeoxynucleotides (CpG-ODNs) in immune cells have been widely studied, little is known regarding their molecular effects in TLR9-positive tumor cells. To better understand the role of these bacterial motifs in cancer cells, we analyzed proteome modifications induced in TLR9-positive tumor cells in vitro and in vivo after CpG-ODN treatment in a rat colon carcinoma model. Proteomics analysis of tumor cells by two-dimensional gel electrophoresis followed by mass spectrometry identified several proteins modulated by bacterial CpG motifs. Among them, several are related to autophagy including potential autophagic substrates. In addition, we observed an increased glyceraldehyde-3-phosphate dehydrogenase expression, which has been shown to be sufficient to trigger an autophagic process. Autophagy is a self-digestion pathway whereby cytoplasmic material is sequestered by a structure termed the autophagosome for subsequent degradation and recycling. As bacteria are known to trigger autophagy, we assessed whether bacterial CpG motifs might induce autophagy in TLR9-positive tumor cells. We showed that CpG-ODN can induce autophagy in rodent and human tumor cell lines and was TLR9-dependent. In addition, an increase in the number of autophagosomes can also be observed in vivo after CpG motif intratumoral injection. Our findings bring new insights on the effect of bacterial CpG motifs in tumor cells and may be relevant for cancer treatment and more generally for gene therapy approaches in TLR9-positive tissues.     

Bacterial models for tumor development. Mini-review

The tumor-inducing effects of Agrobacterium, Bartonella and Helicobacter bacterial species are compared step by step. An analogy for the existence of these individual steps is considered in connection with the development of cancer. The transformations of eukaryotic cells occur in particular in the type IV secretion system, i.e. involving the simultaneous transmission of DNA and protein from bacterial cells to eukaryotic cells. Thus, transfected cells facilitate the indefinite growth of tissue cells and additionally produce growth factors, triggering further bacterial multiplication. The higher numbers of bacteria then produce more transfection and the cycle repeats as long as the host lives. The main limiting factor is the frequency of bacterial infection, while the secondary rate-limiting factors are the levels of transforming growth factors and factors triggering bacteria growth. CONCLUSIONS: Analogous processes are probably responsible for the tumor induction by the three different bacterial species; however, the critical points for eradication are different. The early eradication or limitation of B. henselae or H. pylori can prevent hemangiomas, stomach cancer and malignant cell proliferation. The crown gall formation by A. tumefaciens can only be avoided by prevention of the transforming activity of a single bacterial infection. Questions arise as to what is common in the three processes, and the nature of the rate-limiting step in the three different models. The frequency of transformation is the rate-limiting step, but the co-transmission of the DNA-protein complex is common in the three systems.     

Coupling Between Rates of Bacterial Production and the Abundance of Metabolically Active Bacteria in Lakes, Enumerated Using CTC Reduction and Flow Cytometry

Abstract In natural bacterioplankton assemblages, only a fraction of the total cell count is active, and, therefore, rates of bacterial production should be more strongly correlated to the number of active cells than to the total number of bacteria. However, this hypothesis has seldom been tested. Herein we explore the relationship between rates of bacterial production (measured as leucine uptake) and the number of active bacteria in 14 lakes in southern Québec. Active bacteria are defined as those cells capable of reducing the tetrazolium salt CTC to its fluorescent formazan; these cells were enumerated using flow cytometry. Bacterial production varied two orders of magnitude in the lakes studied, as did the number of active bacteria, whereas the total number of bacteria varied by only sixfold. The number and proportion of active bacteria were similar among lake strata, but rates of bacterial production were highest in the epilimnion and lowest in the hypolimnion. As expected, bacterial production was better correlated to the number of active cells, and bacterial growth rates calculated for active cells ranged from 0.7 to 1.8 day⁻¹, on average threefold higher than those calculated on the basis of total bacterial abundance. Growth rates scaled to active cells were, on average, similar among lake strata and did not show any pattern along a gradient of increasing chlorophyll concentration, so there was no systematic change of bacterial growth rates with lake productivity. In contrast, growth rates scaled to the entire bacterial assemblage were positively correlated to chlorophyll, were tenfold more variable among lakes than growth rates of active cells, and showed larger differences among lake strata. Scaling bacterial production to either the total number or the number of active cells thus results in very different patterns in bacterial growth rates among aquatic systems. Received: 12 July 1996; Accepted: 24 September 1996     

Bacterial age distribution in soil – Generational gaps in adjacent hot and cold spots

Resource patchiness and aqueous phase fragmentation in soil may induce large differences local growth conditions at submillimeter scales. These are translated to vast differences in bacterial age from cells dividing every thirty minutes in close proximity to plant roots to very old cells experiencing negligible growth in adjacent nutrient poor patches. In this study, we link bacterial population demographics with localized soil and hydration conditions to predict emerging generation time distributions and estimate mean bacterial cell ages using mechanistic and heuristic models of bacterial life in soil. Results show heavy-tailed distributions of generation times that resemble a power law for certain conditions, suggesting that we may find bacterial cells of vastly different ages living side by side within small soil volumes. Our results imply that individual bacteria may exist concurrently with all of their ancestors, resulting in an archive of bacterial cells with traits that have been gained (and lost) throughout time–a feature unique to microbial life. This reservoir of bacterial strains and the potential for the reemergence of rare strains with specific functions may be critical for ecosystem stability and function.     

繁茂膜海绵细胞内微生物多样性的研究

采用海绵组织离散、细胞分离的方法,对繁茂膜海绵细胞进行纯化、胞内微生物DNA提取,构建了繁茂膜海绵细胞内微生物的16SrDNA克隆,对其遗传多样性进行了分析,发现海绵细胞内微生物16SrDNA序列主要归类于紫硫细菌门(Proteobacteria)中的α-亚门、γ-亚门和浮霉菌门(Planctomycetes)等类群。与研磨直接提取海绵组织DNA所得海绵组织中总微生物多样性相比,海绵细胞内存在丰富的浮霉菌(23%),说明浮霉菌主要存在于海绵细胞胞内。     

Fimbriae mediated nonspecific adhesion of Salmonella typhimurium to mineral particles

The adhesion of cells of Salmonella typhimurium to albite, biotite, felspar, magnetite and quartz was correlated to the presence of fimbriae and degree of hydrophobicity and charge of the bacterial surface. It was found that the presence of fimbriae resulted in a higher degree of adhesion compared to adhesion of nonfimbriated cells. The significance of the physico-chemical characteristics of fimbriae was shown by a direct linearity between high hydrophobicity of fimbriated cells and degree of adhesion to the mineral particles. Fimbriated cells exhibited higher negative as well as positive surface charge as compared to nonfimbriated cells. Adhesion to several of the minerals was shown to be independent of the extent of negative charges on the bacterial surfaces. A high degree of adhesion to biotite, possibly due to a combination of characteristics of the particles, was not related to either bacterial fimbriation or a physico-chemical characteristic of the bacterial surface. The results of the nonspecific adhesion observed are discussed in terms of available binding sites and distribution of physico-chemical characteristics on the bacterial cell surface structures.