Influence of Fluid Shear and Microbubbles on Bacterial Detachment from a Surface

Prevention of microbial adhesion and detachment of adhering microorganisms from surfaces is important in many environmental, industrial, and medical applications. Fluid shear is an obvious parameter for stimulating microbial detachment from surfaces, but recently it has been pointed out that a passing air-liquid interface also has potential in stimulating microbial detachment. In the present study, the ability of microbubbles to stimulate detachment of bacterial strains from a glass surface is compared with the effects of fluid flow. Adhesion and detachment of Actinomyces naeslundii T14V-J1, Streptococcus oralis J22, and their coadhering aggregates were studied on glass, mounted in a parallel plate flow chamber. High fluid wall shear rates (11,000 to 16,000 s⁻¹) were established in a laminar flow regime in the absence and presence of microbubbles. Wall shear rates stimulated detachment ranging from 70% to 30% for S. oralis and A. naeslundii, respectively. Coadhering aggregates were detached up to 54%. The presence of microbubbles in the flow increased the detachment of A. naeslundii within 2 min of flow from 40% in the absence of microbubbles to 98%, while detachment of neither S. oralis nor coadhering aggregates was affected by the presence of microbubbles. In summary, extremely high fluid flows can be effective in stimulating microbial detachment, depending on the strain involved. The addition of microbubbles to the flow allows the detachment of tenaciously adhering bacteria not detached by flow alone, but not of adhering coaggregates.     

Erosion from Staphylococcus aureus Biofilms Grown under Physiologically Relevant Fluid Shear Forces Yields Bacterial Cells with Reduced Avidity to Collagen

An estimated 65% of infective diseases are associated with the presence of bacterial biofilms. Biofilm-issued planktonic cells promote blood-borne, secondary sites of infection by the inoculation of the infected sites with bacteria from the intravascular space. To investigate the potential role of early detachment events in initiating secondary infections, we studied the phenotypic attributes of Staphylococcus aureus planktonic cells eroding from biofilms with respect to expression of the collagen adhesin, CNA. The collagen-binding abilities of S. aureus have been correlated to the development of osteomyelitis and septic arthritis. In this study, we focused on the impact of CNA expression on S. aureus adhesion to immobilized collagen in vitro under physiologically relevant shear forces. In contrast to the growth phase-dependent adhesion properties characteristic of S. aureus cells grown in suspension, eroding planktonic cells expressed invariant and lower effective adhesion rates regardless of the age of the biofilm from which they originated. These results correlated directly with the surface expression level of CNA. However, subsequent analysis revealed no qualitative differences between biofilms initiated with suspension cells and secondary biofilms initiated with biofilm-shed planktonic cells. Taken together, our findings suggest that, despite their low levels of CNA expression, S. aureus planktonic cells shed from biofilms retain the capacity for metastatic spread and the initiation of secondary infection. These findings demonstrate the need for a better understanding of the phenotypic properties of eroding planktonic cells, which could lead to new therapeutic strategies to target secondary infections.     

Rumen Bacterial and Protozoal Responses to Insecticide Substrates

Insecticides containing organophosphate, chlorinated hydrocarbon, and carbamate were tested with bovine ruminal ingesta fractions. Rumen bacteria exposed to insecticide levels of 0 to 500 ppm in rumen fluid for 4 hr were inoculated into rumen fluid-starch feed extract medium. No apparent significant bacterial count inhibitions were noted. Also, when insecticides were used as carbon sources at concentrations of 500 ppm in carbohydrate-limited media, no increases in bacterial counts were indicated. Warburg manometric data showed that paraffin oil-Triton X-155 preparations of dimethoate, Diazinon, lindane, Thiodan and Sevin stimulated gas production in holotrich protozoa. Entodinium simplex, an oligotrich, produced less gas with insecticide substrates per unit of dry weight than did an Isotricha sp. Rumen bacteria and plant debris fractions from ruminal ingesta provided with insecticides did not give increased manometric responses over the endogenous control vessels. Washed suspensions of I. intestinalis produced volatile fatty acids in excess of the endogenous suspensions when provided insecticide substrates. Thiodan dissimilation by I. intestinalis was followed colorimetrically with 15% loss in substrate in 1 hr of incubation at 39 C. Diazinon-C¹⁴ substrate uptake was demonstrated with suspensions of E. simplex and I. intestinalis. Rumen ciliates are suggested as a possible means for screening out useful insecticides susceptible to microbial dissimilation for use on forage and other cattle-feed crops.     

Bacterial Activity Associated with the Decomposition of Woody Substrates in a Stream Sediment

Ground bark and heartwood from Alnus rubra and Pseudotsuga menziesii were added to a muddy sediment from a small Oregon stream and incubated in situ. Carbon dioxide and methane production rates were increased by all amendments, the biggest increase being shown with A. rubra wood. Except for sediment amended with A. rubra wood, nitrogen fixation rates from all treatments (including the control) were approximately 0.1 nmol/g per h throughout the 6-month study period. Contrary to expectations, neither bark had a noticeable adverse effect on microbial activity, but the A. rubra wood promoted nitrogen fixation. These results help to explain the faster rate of decomposition of A. rubra wood in water compared with that of P. menziesii described in the literature. The uptake kinetics of glucose (V_max) did not follow the same pattern as gas evolution.     

Bacterial adaptation: Macromolecular biosynthesis during diauxic growth of Escherichia coli

Abstract Synthesis of DNA, RNA and protein was studied during a diauxic adaptation (glucose- to acetate-utilization) of Escherichia coli B. The multiphasic transition described illuminates certain features of the mechanisms regulating metabolism of the 3 macromolecules involved in the genetic flow of information.     

Quantitative Determination of Dye Uptake by Bacterial Cells

A colorimetric method is presented for the quantitative determination of dye uptake by bacterial cells. Experiments showed that the dye to cell ratio was of major importance in controlling the amount of dye taken up per weight of bacterial cells. Approximate dye saturation of cells could be obtained (at pH 6.1 to 6.3) although the dye uptake curves did not absolutely level off.     

Increased Placental Growth Factor in Cerebrospinal Fluid of Patients with Epilepsy

Recent studies suggest that angiogenesis and vascular endothelial growth factor (VEGF) are involved in the pathophysiology of epilepsy. However, relatively little data are available linking placenta growth factor (PIGF) with epilepsy. In this study, we assessed concentrations of PIGF in cerebrospinal fluid (CSF) of 60 epileptic patients and 24 non-seizure subjects using sandwich enzyme-linked immunosorbent assays. Epileptic patients in general had higher concentration of CSF-PIGF than controls (7.95 ± 0.88 ng/l vs. 5.87 ± 0.79 ng/l, P < 0.01). CSF-PIGF level in secondary epileptic patients (8.59 ± 1.26 ng/l) was higher than that in idiopathic epileptic patients (7.62 ± 0.20 ng/l) (P < 0.05). In idiopathic epilepsy, CSF-PIGF level in patients with high seizure frequency was higher than those in patients with low seizure frequency and seizure-free in recent 3 years (7.78 ± 0.23 ng/l vs. 7.49 ± 0.09 ng/l and 7.59 ± 0.10 ng/l, P < 0.05). Concentration of CSF-PIGF in patients with a disease duration of > 5 years was higher than those in patients with durations of 1-5 years and <1 year (7.72 ± 0.20 ng/l vs. 7.52 ± 0.09 ng/l and 7.41 ± 0.07 ng/l, P < 0.05). These results indicate that preexisting brain damage, seizure frequency and disease duration are important factors contributing to elevated PIGF.     

Oxidative Stress in Cerebrospinal Fluid of Patients with Aseptic and Bacterial Meningitis

This study aimed to determine whether patients with aseptic and bacterial meningitis presented alterations in oxidative stress parameters of cerebrospinal fluid (CSF). A total of 30 patients were used in the research. The CSF oxidative stress status has been evaluated through many parameters, such as lipid peroxidation through thiobarbituric acid reactive substances (TBARS) and antioxidant defense systems such as superoxide dismutase (SOD), glutathione S-transferase (GST), reduced glutathione (GSH) and ascorbic acid. TBARS levels, SOD and GST activity increase in aseptic meningitis and in bacterial meningitis. The ascorbic acid concentration increased significantly in patients with both meningitis types. The reduced glutathione levels were reduced in CSF of patients with aseptic and bacterial meningitis. In present study we may conclude that oxidative stress contributes at least in part to the severe neurological dysfunction found in meningitis.     

Breakdown of Diazotized Proteins and Synthetic Substrates by Rumen Bacterial Proteases

Several different kinds of substrate were used to investigate the proteolytic activity of rumen bacteria and of proteases released from rumen bacteria by blending (“coat proteases”). These substrates included diazotized feed proteins and diazotized soluble and insoluble pure proteins. It was concluded that, while solubility was an important factor, the secondary and tertiary structure of a protein had a major influence on its rate of digestion. The resistance of elastin congo red to digestion indicated that similar fibrous proteins in plant material might resist proteolytic attack by rumen bacteria. Coat proteases had a broad specificity, including several exo- and endopeptidase activities, as determined by using synthetic peptide substrates.     

Increased Potency and Longevity of Gene Silencing Using Validated Dicer Substrates

Chemically synthesized small interfering RNAs (siRNAs) are tools used for silencing the expression of a single gene. They are mainly employed in basic research applications, but may also have great potential in therapeutic applications. Longer double-stranded RNAs, such as Dicer-substrate 27mers, trigger gene silencing through the intrinsic RNAi pathway. The design of these Dicer-substrate 27mers has been optimized so they can be oriented by Dicer to consistently select the antisense (guide) strand after cleavage to shorter siRNAs, leading to predictable mRNA cleavage. In this paper we describe evidence that these Dicer-substrate 27mers produce more potent and sustained gene silencing for four genes when compared with synthetic 21mers that have the same guide-strand sequence. Furthermore, improved silencing by these 27mers is often more pronounced at lower concentrations.     

细菌铁离子利用系统与宿主抗感染免疫

铁离子对于绝大多数微生物及其宿主都是必需的营养物质,它是许多蛋白和酶的重要辅助因子。致病菌为了成功致病,进化出了多种机制来摄取宿主体内的铁离子,其中主要包括三价铁离子转运系统和亚铁血红素转运系统。而对于宿主而言,铁离子虽然在细胞呼吸和DNA复制等过程中扮演着重要的角色,但过多的铁离子也会产生细胞毒性。因此,宿主体内的铁离子浓度必须受到严格的调控。为限制病原菌感染,宿主先天性免疫系统进化出一系列限制自身铁离子进入微生物的机制,这一过程被称为宿主的"营养免疫"。从病原菌和宿主两个方面详细讨论病原菌是如何从宿主获取铁离子以及宿主如何防止细菌获取铁离子的分子机制,能为更好地提高宿主免疫力来阻止细菌感染和开发有效的非抗生素类药物提供理论依据。     

Mechanisms and Significance of the Increased Brain Uptake of Tryptophan

Changes in brain tryptophan concentrations may affect the synthesis of brain serotonin (5-hydroxytryptamine, 5-HT). Concentrations of tryptophan are regulated more than those of any other amino acid. Such stimuli as acute stress, carbohydrate ingestion, and treatment with various drugs increase the brain content of tryptophan. Treatment of rats and mice with interleukin-1 (IL-1), interleukin-6 (IL-6), lipopolysaccharide (LPS), and β-adrenoceptor agonists, as well as a variety of stressors, such as footshock and restraint, all increase brain concentrations of tryptophan. The peak effect following both acute stress and β-adrenoceptor agonist administration occurs within 30–60 min, whereas the peak effect following LPS and the cytokines occurs much later at around 4–8 h. Experiments using the ganglionic blocker chlorisondamine, and β-adrenoceptor antagonists suggest that the sympathetic nervous system plays an important role in the modulation of brain tryptophan concentrations. The mechanisms involved in the increases observed in brain tryptophan are discussed, as well as their possible biological significance. Special issue dedicated to Dr. Simo S. Oja     

Fluid Shear Stress Increases Neutrophil Activation via Platelet-Activating Factor

Leukocyte exposure to hemodynamic shear forces is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. G-protein coupled receptors on neutrophils, which bind to chemoattractants and play a role in neutrophil chemotaxis, have been implicated as fluid shear stress sensors that control neutrophil activation. Recently, exposure to physiological fluid shear stresses observed in the microvasculature was shown to reduce neutrophil activation in the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine. Here, however, human neutrophil preexposure to uniform shear stress (0.1–2.75 dyn/cm²) in a cone-and-plate viscometer for 1–120 min was shown to increase, rather than decrease, neutrophil activation in the presence of platelet activating factor (PAF). Fluid shear stress exposure increased PAF-induced neutrophil activation in terms of L-selectin shedding, α_Mβ₂ integrin activation, and morphological changes. Neutrophil activation via PAF was found to correlate with fluid shear stress exposure, as neutrophil activation increased in a shear stress magnitude- and time-dependent manner. These results indicate that fluid shear stress exposure increases neutrophil activation by PAF, and, taken together with previous observations, differentially controls how neutrophils respond to chemoattractants.     

Fluid Shear Stress Increases Neutrophil Activation via Platelet-Activating Factor

Leukocyte exposure to hemodynamic shear forces is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. G-protein coupled receptors on neutrophils, which bind to chemoattractants and play a role in neutrophil chemotaxis, have been implicated as fluid shear stress sensors that control neutrophil activation. Recently, exposure to physiological fluid shear stresses observed in the microvasculature was shown to reduce neutrophil activation in the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine. Here, however, human neutrophil preexposure to uniform shear stress (0.1–2.75 dyn/cm²) in a cone-and-plate viscometer for 1–120 min was shown to increase, rather than decrease, neutrophil activation in the presence of platelet activating factor (PAF). Fluid shear stress exposure increased PAF-induced neutrophil activation in terms of L-selectin shedding, α_Mβ₂ integrin activation, and morphological changes. Neutrophil activation via PAF was found to correlate with fluid shear stress exposure, as neutrophil activation increased in a shear stress magnitude- and time-dependent manner. These results indicate that fluid shear stress exposure increases neutrophil activation by PAF, and, taken together with previous observations, differentially controls how neutrophils respond to chemoattractants.     

Separation of Bacterial Ribosomal Ribonucleic Acid from Its Macromolecular Precursors by Polyacrylamide Gel Electrophoresis

Electrophoresis on polyacrylamide gels was found to be a powerful technique for separating the mature from the precursor forms of bacterial ribosomal nucleic acid (rRNA). The separation of the 16S rRNA from its precursor was, for all practical purposes, complete; that of the 23S rRNA from its precursor was detectable but incomplete. When mature and precursor rRNA preparations were heated to randomize secondary structure, etc., and then cooled, it was found that electrophoretic mobility differences between mature forms of rRNA and their precursors persisted. This, in conjunction with the rather large electrophoretic mobility differences between mature and precursor forms, can be taken as strong evidence for a molecular weight difference between mature rRNA and its precursor forms of RNA. With the 16S rRNA, this difference could be as large as 130,000 daltons.     

Bacterial DNA Uptake Sequences Can Accumulate by Molecular Drive Alone

Uptake signal sequences are DNA motifs that promote DNA uptake by competent bacteria in the family Pasteurellaceae and the genus Neisseria. The genomes of these bacteria contain many copies of their canonical uptake sequence (often >100-fold overrepresentation), so the bias of the uptake machinery causes cells to prefer DNA derived from close relatives over DNA from other sources. However, the molecular and evolutionary forces responsible for the abundance of uptake sequences in these genomes are not well understood, and their presence is not easily explained by any of the current models of the evolution of competence. Here we describe use of a computer simulation model to thoroughly evaluate the simplest explanation for uptake sequences, that they accumulate in genomes by a form of molecular drive generated by biased DNA uptake and evolutionarily neutral (i.e., unselected) recombination. In parallel we used an unbiased search algorithm to characterize genomic uptake sequences and DNA uptake assays to refine the Haemophilus influenzae uptake specificity. These analyses showed that biased uptake and neutral recombination are sufficient to drive uptake sequences to high densities, with the spacings, stabilities, and strong consensuses typical of uptake sequences in real genomes. This result greatly simplifies testing of hypotheses about the benefits of DNA uptake, because it explains how genomes could have passively accumulated sequences matching the bias of their uptake machineries.MANY bacteria are able to take up DNA fragments from their environment, a genetically specified trait called natural competence (Chen and Dubnau 2004; Johnsborg et al. 2007; Maughan et al. 2008). Many other species have homologs of competence genes, suggesting that although they are not competent under laboratory conditions, they may be competent under natural conditions (Claverys and Martin 2003; Kovacs et al. 2009). Such a widespread trait must be beneficial but the evolutionary function of DNA uptake remains controversial. Cells can use the nucleotides released by degradation of both incoming DNA and any strands displaced by its recombination, thus reducing demands on their nucleotide salvage and biosynthesis pathways (Redfield 1993; Palchevskiy and Finkel 2009). Cells may also benefit if recombination of the incoming DNA provides templates for DNA repair or introduces beneficial mutations, but may suffer if recombination introduces damage or harmful mutations (Redfield 1988; Michod et al. 2008).Although most bacteria that have been tested show no obvious preferences for specific DNA sources or sequences, bacteria in the family Pasteurellaceae and the genus Neisseria strongly prefer DNA fragments from close relatives. Two factors are responsible: First, the DNA uptake machineries of these bacteria are strongly biased toward certain short DNA sequence motifs. Second, the genomes of these bacteria contain hundreds of occurrences of the preferred sequences. The Pasteurellacean motif is called the uptake signal sequence (USS); its Neisseria counterpart is called the DNA uptake sequence (DUS). All Neisseria genomes contain the same consensus DUS [core GCCGTCTGAA (Treangen et al. 2008)], but divergence in the Pasteurellaceae has produced two subclades, one of species sharing the canonical Haemophilus influenzae 9-bp USS (Hin-USS core AAGTGCGGT) and the other of species with a variant USS that differs at three core positions (Apl-USS core: ACAAGCGGT) and has a longer flanking consensus (Redfield et al. 2006). Uptake sequence biases are strong but not absolute; for example, replacing the Hin-USS with the Apl-USS reduces H. influenzae DNA uptake only 10-fold (Redfield et al. 2006) and DNA from Escherichia coli is taken up in the absence of competing H. influenzae DNA (Goodgal and Mitchell 1984).Most studies of the distribution and consensus of uptake sequences in genomes have examined only those occurrences that perfectly match the above core DUS and USS sequences. Here we call these perfect matches “core-consensus” (cc) uptake sequences. These cc-uptake sequences occupy ∼1% of their genomes; they are equally frequent in the plus and minus orientations of the genome sequence but are underrepresented in coding sequences, with the noncoding 14% and 20% of their respective genomes containing 35% of cc-USSs and 65% of cc-DUSs (Smith et al. 1995). Although many of these intergenic cc-DUSs and cc-USSs occur in inverted-repeat pairs that function as terminators (Kingsford et al. 2007), most uptake sequences are too far apart or in genes or other locations where termination does not occur. Within coding regions uptake sequences occur more often in weakly conserved genes, in weakly conserved parts of genes, and in reading frames that encode common tripeptides (Findlay and Redfield 2009), all of which are consistent with selection acting mainly to eliminate mutations that improve uptake from genome regions constrained by coding or other functions.Analyses that focus on cc-uptake sequences effectively treat uptake sequences as replicative elements (Smith et al. 1995; Redfield et al. 2006; Ambur et al. 2007; Treangen et al. 2008). However, USS and DUS are known to originate in situ by normal mutational processes, mainly point mutations, and to spread between genomes mainly by homologous recombination (Redfield et al. 2006; Treangen et al. 2008). As they are not replicating elements, why are they up to 1000-fold more common in their genomes than expected for unselected sequences (e.g., H. influenzae, 1471 cc-USS vs. 8 expected by chance; N. gonorrheae, 1892 cc-DUS vs. 2 expected by chance)?The explanation for this abundance must lie with the specificity of the DNA uptake system, because the strong correspondence between the uptake bias and the uptake sequences in the genome is far too improbable to be a coincidence. However, uptake specificity is not easily accommodated by either of the hypothesized functions of DNA uptake. If bacteria take up DNA to get benefits from homologous genetic recombination, the combination of uptake bias and uptake sequences might serve as a mate-choice adaptation that maximizes these benefits by excluding foreign DNAs (Treangen et al. 2008). Although this explanation is appealing, it requires simultaneous evolution of bias in the uptake machinery and of genomic sequences matching this bias. Another problem is that the genomic sequences can be “selected” only after the cell carrying them is dead. On the other hand, if bacteria instead take up DNA as a source of nutrients, all DNAs should be equally useful, so uptake bias and uptake sequences would likely reduce rather than increase this benefit. Although the sequence bias could be explained as a consequence of mechanistic constraints on DNA uptake, this would not account for the high density of the preferred sequences in the genome.However, both hypotheses may be simplified by a process called molecular drive, under which uptake sequences would gradually accumulate over evolutionary time as a direct consequence of biased uptake and recombination (Danner et al. 1980; Bakkali et al. 2004; Bakkali 2007), without any need for natural selection. This drive is proposed to work as follows: First, random mutation continuously creates variation in DNA sequences that affects their probability of uptake, and random cell death allows DNA fragments containing preferred variants to be taken up by other cells. Second, repeated recombination of such preferred DNA sequences with their chromosomal homologs gradually increases their abundance in the genomes of competent cells'' descendants. Thus mutations that create matches to the bias of the uptake machinery are horizontally transmitted to other members of the same species more often than other mutations. Because some recombination may be inevitable even if DNA''s main benefit is nutritional, molecular drive could account for uptake sequence accumulation under both hypotheses, leaving only the biased uptake process to be explained by natural selection for either genetic variation or nutrients.Although drive is plausible, its ability to account for the observed properties of genomic uptake sequences has never been evaluated. To do this, we developed a realistic computer simulation model that includes only the processes thought to generate molecular drive. Below we first use this model to identify the conditions that determine whether uptake sequences will accumulate and then compare the properties of these simulated uptake sequences to those of the uptake sequences in the N. meningitidis and H. influenzae genomes. In parallel we use unbiased motif searches to better characterize genomic uptake sequences and DNA uptake assays to refine the H. influenzae uptake specificity.