In Situ Analysis of Nitrifying Biofilms as Determined by In Situ Hybridization and the Use of Microelectrodes

We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in domestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these techniques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In situ hybridization revealed that bacteria belonging to the genus Nitrosomonas were the numerically dominant ammonia-oxidizing bacteria in both types of biofilms. Bacteria belonging to the genus Nitrobacter were not detected; instead, Nitrospira-like bacteria were the main nitrite-oxidizing bacteria in both types of biofilms. Nitrospira-like cells formed irregularly shaped aggregates consisting of small microcolonies, which clustered around the clusters of ammonia oxidizers. Whereas most of the ammonia-oxidizing bacteria were present throughout the biofilms, the nitrite-oxidizing bacteria were restricted to the active nitrite-oxidizing zones, which were in the inner parts of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of nitrite-oxidizing bacteria, as determined by FISH.     

In Vitro Laser Ablation of Natural Marine Biofilms

We studied the efficiency of pulsed low-power laser irradiation of 532 nm from an Nd:YAG (neodymium-doped yttrium-aluminum-garnet) laser to remove marine biofilm developed on titanium and glass coupons. Natural biofilms with thicknesses of 79.4 ± 27.8 μm (titanium) and 107.4 ± 28.5 μm (glass) were completely disrupted by 30 s of laser irradiation (fluence, 0.1 J/cm²). Laser irradiation significantly reduced the number of diatoms and bacteria in the biofilm (paired t test; P < 0.05). The removal was better on titanium than on glass coupons.     

Quorum-Sensing Mutations Affect Attachment and Stability of Burkholderia cenocepacia Biofilms

Biofilm formation in Burkholderia cenocepacia has been shown to rely in part on acylhomoserine lactone-based quorum sensing. For many other bacterial species, it appears that both the initial adherence and the later stages of biofilm maturation are affected when quorum sensing pathways are inhibited. In this study, we examined the effects of mutations in the cepIR and cciIR quorum-sensing systems of Burkholderia cenocepacia K56-2 with respect to biofilm attachment and antibiotic resistance. We also examined the role of the cepIR system in biofilm stability and structural development. Using the high-throughput MBEC assay system to produce multiple equivalent biofilms, the biomasses of both the cepI and cepR mutant biofilms, measured by crystal violet staining, were less than half of the value observed for the wild-type strain. Attachment was partially restored upon providing functional gene copies via multicopy expression vectors. Surprisingly, neither the cciI mutant nor the double cciI cepI mutant was deficient in attachment, and restoration of the cciI gene resulted in less attachment than for the mutants. Meanwhile, the cciR mutant did show a significant reduction in attachment, as did the cciR cepIR mutant. While there was no change in antibiotic susceptibility with the individual cepIR and cciIR mutants, the cepI cciI mutant biofilms were more sensitive to ciprofloxacin. A significant increase in sensitivity to removal by sodium dodecyl sulfate was seen for the cepI and cepR mutants. Flow cell analysis of the individual cepIR mutant biofilms indicated that they were both structurally and temporally impaired in attachment and development. These results suggest that biofilm structural defects might be present in quorum-sensing mutants of B. cenocepacia that affect the stability and resistance of the adherent cell mass, providing a basis for future studies to design preventative measures against biofilm formation in this species, an important lung pathogen of cystic fibrosis patients.     

Quorum-Sensing Genes in Pseudomonas aeruginosa Biofilms: Their Role and Expression Patterns

Acylated homoserine lactone molecules are used by a number of gram-negative bacteria to regulate cell density-dependent gene expression by a mechanism known as quorum sensing (QS). In Pseudomonas aeruginosa, QS or cell-to-cell signaling controls expression of a number of virulence factors, as well as biofilm differentiation. In this study, we investigated the role played by the las and rhl QS systems during the early stages of static biofilm formation when cells are adhering to a surface and forming microcolonies. These studies revealed a marked difference in biofilm formation between the PAO1 parent and the QS mutants when glucose, but not citrate, was used as the sole carbon source. To further elucidate the contribution of lasI and rhlI to biofilm maturation, we utilized fusions to unstable green fluorescent protein in concert with confocal microscopy to perform real-time temporal and spatial studies of these genes in a flowing environment. During the course of 8-day biofilm development, lasI expression was found to progressively decrease over time. Conversely, rhlI expression remained steady throughout biofilm development but occurred in a lower percentage of cells. Spatial analysis revealed that lasI and rhlI were maximally expressed in cells located at the substratum and that expression decreased with increasing biofilm height. Because QS was shown previously to be involved in biofilm differentiation, these findings have important implications for the design of biofilm prevention and eradication strategies.     

In Vitro and In Situ Absorption of Methionine Sulfoxide in Rat Small Intestine

Absorption of methionine and its sulfoxide was investigated in vitro with everted sacs and in situ with circulated loops of rat small intestine. Transmural transport and tissue accumulation of methionine sulfoxide in the everted sacs were in fair agreement with those of methionine. Apparent kinetic parameters for the difference of transmural transport in the absence and presence of 10^?5 m carbonylcyanide m-chlorophenylhydrazone, i.e. for the energy-dependent active transport, were similar for both methionine and its sulfoxide. Methionine was found at a low level in the serosal fluid of the everted sac on incubation with methionine sulfoxide. It was attributed to the methionine leaked out from the tissue but not to that formed by reduction of methionine sulfoxide during the course of intestinal transport. Similar transport was also observed in situ in circulated intestinal loops for methionine and its sulfoxide. The absorption efficiency of methionine sulfoxide in the small intestine is not the reason for the decreased nutritional availability of the most likely oxidation product of methionine.     

Model System for Growing and Quantifying Streptococcus pneumoniae Biofilms In Situ and in Real Time

Streptococcus pneumoniae forms biofilms, but little is known about its extracellular polymeric substances (EPS) or the kinetics of biofilm formation. A system was developed to enable the simultaneous measurement of cells and the EPS of biofilm-associated S. pneumoniae in situ over time. A biofilm reactor containing germanium coupons was interfaced to an attenuated total reflectance (ATR) germanium cell of a Fourier transform infrared (FTIR) laser spectrometer. Biofilm-associated cells were recovered from the coupons and quantified by total and viable cell count methods. ATR-FTIR spectroscopy of biofilms formed on the germanium internal reflection element (IRE) of the ATR cell provided a continuous spectrum of biofilm protein and polysaccharide (a measure of the EPS). Staining of the biofilms on the IRE surface with specific fluorescent probes provided confirmatory evidence for the biofilm structure and the presence of biofilm polysaccharides. Biofilm protein and polysaccharides were detected within hours after inoculation and continued to increase for the next 141 h. The polysaccharide band increased at a substantially higher rate than did the protein band, demonstrating increasing coverage of the IRE surface with biofilm polysaccharides. The biofilm total cell counts on germanium coupons stabilized after 21 h, at approximately 10⁵ cells per cm², while viable counts decreased as the biofilm aged. This system is unique in its ability to detect and quantify biofilm-associated cells and EPS of S. pneumoniae over time by using multiple, corroborative techniques. This approach could prove useful for the study of biofilm processes of this or other microorganisms of clinical or industrial relevance.     

In Situ Activity and Spatial Organization of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria in Biofilms

We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that “Brocadia”-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 μm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH₄⁺ and NO₂⁻ consumption rates decreased from 0.68 and 0.64 μmol cm⁻² h⁻¹ at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 μmol cm⁻² h⁻¹ at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH₄⁺ and NO₂⁻ and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O₂ or organic compounds, which consequently established suitable microenvironments for anammox bacteria.     

Comparison of In Situ and In Vitro Rates of Methane Release in Freshwater Sediments

Anaerobic lake sediment incubated in vitro was investigated for its ability to mimic natural in situ sediment activities, using rate of methane production for the comparison. Two lakes with different rates and seasonal patterns of methanogenic activity were compared. There was good agreement (at the 97.5% confidence level) between rates of in situ methane release and initial (lasting an average of 120 h) rates of production measured in vitro in surface (0- to 3-cm) sediment. Evidence from this study, and others, indicated that it is the in situ surface sediment methane production which is primarily responsible for maintaining in situ methane release, and thus the above agreement was what was expected if surface in situ activity was maintained in vitro. When deeper sediment was investigated, however, the sum of in vitro rates from 0 to 20 cm (measured in 1.5- to 3-cm intervals) was much higher than in situ release rates and would have resulted in an impossibly high volume of gas. The extra gas could not have been stored within the sediments. We conclude that the in situ methanogenic activity of the 0- to 3-cm anaerobic surface sediments could be preserved during removal and laboratory incubation. However, similar treatment of deeper sediment appeared to stimulate methanogenic activity.     

In Situ and In Vitro Expression of Protein Kinase C Alpha in Human Melanocytes

Protein kinase C (PKC) is a multigene family of at least 12 isoforms involved in the transduction of extracellular signals. We investigated whether PKC-α, a major isoform known to be relatively abundant in brain tissue, is increased in human melanocytes relative to keratinocytes in vitro and in situ. Immunohistochemical staining for PKC-α in frozen neonatal human foreskin exhibited intermittent 2–3+ staining along the basal cell layer consistent with melanocytes, and 0–1+ staining of keratinocytes (on a scale of 0–3). Microscopic densitometry of the intermittent cellular staining was at least 3-fold greater than that of adjacent keratinocyte cell cytoplasm. Sequential frozen sections revealed similar intermittent cell staining with PKC-α and Mel-5 (tyrosinase related protein-1), known to specifically react with melanocytes. Northern blot analysis with a specific cDNA probe for PKC-α showed strong PKC-α mRNA expression in cultured melanocytes, whereas PKC-α mRNA in cultured non-stratifying keratinocytes was expressed at low levels. Western blot analysis revealed a prominent PKC-α band at approximately 80 kDa in melanocytes as opposed to a weak band in keratinocytes. Densitometry of the northern and western blots revealed that melanocytes had at least 10-fold more PKC-α mRNA and approximately 6-fold more PKC-α protein expression than keratinocytes. Total PKC activity measured in vitro revealed that melanocytes had 5-fold more activity than keratinocytes. The marked difference in melanocyte and keratinocyte expression of PKC-α provides further evidence for cell type specificity in the balance of PKC-α expression and may implicate differential PKC isoform signaling pathways in neuro-ectodermally derived cells.     

Francisella novicida Forms In Vitro Biofilms Mediated by an Orphan Response Regulator

Francisella tularensis is associated with water and waterways and infects many species of animals, insects, and protists. The mechanism Francisella utilizes to persist in the environment and in tick vectors is currently unknown. We have demonstrated for the first time that Francisella novicida, a model organism of F. tularensis, forms a biofilm in vitro. Selected F. novicida transposon mutants were tested for their ability to form biofilm compared to the wildtype F. novicida strain. Mutation of the putative qseB gene led to an impairment in the ability to form biofilm with no impairment in bacterial growth. A qseC mutant had impaired growth but demonstrated a marked impairment in biofilm production. Mutation in capC affected both bacterial growth and biofilm formation, but no biofilm production impairment was seen with capB or pilE mutants. A deletion mutant in the orphan response regulator FTN_1465, which we propose is the putative QseB, formed significantly less biofilm than the wildtype. When FTN_1465 was complemented back into the deletion mutant, biofilm formation was restored. Thus, the orphan response regulator FTN_1465 is an important factor in biofilm production in vitro in F. novicida. These results demonstrate that Francisella species are able to form biofilms in vitro, suggesting that biofilm formation may be important for the lifecycle of this organism.     

Three TRH-Like Molecules Are Released from Rat Hypothalamus In Vitro

TRH-like immunoreactivity distinct from TRH is present in various tissues and fluids. In order to determine whether TRH-like molecules are secreted by the hypothalamus, we analyzed tissues and media from hypothalamic slices incubated in Krebs Ringer bicarbonate. Media from basal or high KCl conditions contained 3 TRH-like molecules evidenced by reverse phase high performance liquid chromatography followed by TRH radioimmunoassay. Peak I corresponded to authentic TRH (73% of total immunoreactivity) and peaks II and III had a higher retention time. These additional TRH-like forms were neither detected in hypothalamic tissue nor in tissue or medium from olfactory bulb. Gel filtration analysis of hypothalamic media revealed only one TRH-like peak eluting as TRH, suggesting that the molecular weights of peaks II and III are similar to that of TRH. Peak II retention time was similar to that of pglu-phe-proNH₂. We analysed if they could be produced by post secretory metabolism of TRH. Incubation of hypothalamic slices with [³H-Pro]-TRH did not produce radioactive species comigrating with peaks II or III. However, it induced rapid degradation to [³H-Pro]-his-prodiketopiperazine ([³H]-HPDKP). Inhibitor profile suggested that pyroglutamyl aminopeptidase II, but not pyroglutamyl aminopeptidase I, is responsible for [³H]-HPDKP production. These data are consistent with the hypothesis that pyroglutamyl aminopeptidase II is the main aminopeptidase degrading TRH in hypothalamic extracellular fluid. Furthermore, we suggest that the hypothalamus releases additional TRH-like molecules, one of them possibly pglu-phe-proNH₂, which may participate in control of adenohypophyseal secretions.     

In Situ Prebiotics for Weaning Piglets: In Vitro Production and Fermentation of Potato Galacto-Rhamnogalacturonan

Postweaning diarrhea (PWD) in pigs is a leading cause of economic loss in pork production worldwide. The current practice of using antibiotics and zinc to treat PWD is unsustainable due to the potential of antibiotic resistance and ecological disturbance, and novel methods are required. In this study, an in vitro model was used to test the possibility of producing prebiotic fiber in situ in the gastrointestinal (GI) tract of the piglet and the prebiotic activity of the resulting fiber in the terminal ileum. Soluble fiber was successfully produced from potato pulp, an industrial waste product, with the minimal enzyme dose in a simulated upper GI tract model extracting 26.9% of the initial dry matter. The fiber was rich in galactose and galacturonic acid and was fermented at 2.5, 5, or 10 g/liter in a glucose-free medium inoculated with the gut contents of piglet terminal ileum. Fermentations of 5 g/liter inulin or 5 g/liter of a purified potato fiber were used as controls. The fibers showed high fermentability, evident by a dose-dependent drop in pH and an increase in the organic acid content, with lactate in particular being increased. Deep sequencing showed a significant increase in the numbers of Lactobacillus and Veillonella organisms and an insignificant increase in the numbers of Clostridium organisms as well as a decrease in the numbers of Streptococcus organisms. Multivariate analysis showed clustering of the treatment groups, with the group treated with purified potato fiber being clearly separated from the other groups, as the microbiota composition was 60% Lactobacillus and almost free of Clostridium. For animal studies, a dosage corresponding to the 5-g/liter treatment is suggested.     

In Situ Gene Expression in Mixed-Culture Biofilms: Evidence of Metabolic Interactions between Community Members

Microbial communities growing in laboratory-based flow chambers were investigated in order to study compartmentalization of specific gene expression. Among the community members studied, the focus was in particular on Pseudomonas putida and a strain of an Acinetobacter sp., and the genes studied are involved in the biodegradation of toluene and related aromatic compounds. The upper-pathway promoter (Pu) and the meta-pathway promoter (Pm) from the TOL plasmid were fused independently to the gene coding for the green fluorescent protein (GFP), and expression from these promoters was studied in P. putida, which was a dominant community member. Biofilms were cultured in flow chambers, which in combination with scanning confocal laser microscopy allowed direct monitoring of promoter activity with single-cell spatial resolution. Expression from the Pu promoter was homogeneously induced by benzyl alcohol in both community and pure-culture biofilms, while the Pm promoter was induced in the mixed community but not in a pure-culture biofilm. By sequentially adding community members, induction of Pm was shown to be a consequence of direct metabolic interactions between an Acinetobacter species and P. putida. Furthermore, in fixed biofilm samples organism identity was determined and gene expression was visualized at the same time by combining GFP expression with in situ hybridization with fluorescence-labeled 16S rRNA targeting probes. This combination of techniques is a powerful approach for investigating structure-function relationships in microbial communities.     

Studies on Mefenamic Acid Microparticles: Formulation, In Vitro Release, and In Situ Studies in Rats

In this study, we investigated the in vitro characteristics of mefenamic acid (MA) microparticles as well as their effects on DNA damage. MA-loaded chitosan and alginate beads were prepared by the ionotropic gelation process. Microsponges containing MA and Eudragit RS 100 were prepared by quasi-emulsion solvent diffusion method. The microparticles were characterized in terms of particle size, surface morphology, encapsulation efficiency, and in vitro release profiles. Most of the formulation variables manifested an influence on the physical characteristics of the microparticles at varying degrees. We also studied the effects of MA, MA-loaded microparticles, and three different polymers on rat brain cortex DNA damage. Our results showed that DNA damage was higher in MA-loaded Eudragit microsponges than MA-loaded biodegradable chitosan or alginate microparticles.     

Nanosuspension Based In Situ Gelling Nasal Spray of Carvedilol: Development, In Vitro and In Vivo Characterization

The objective of the present investigation was to develop in situ gelling nasal spray formulation of carvedilol (CRV) nanosuspension to improve the bioavailability and therapeutic efficiency. Solvent precipitation–ultrasonication method was opted for the preparation of CRV nanosuspension which further incorporated into the in situ gelling polymer phase. Optimized formulation was extensively characterized for various physical parameters like in situ gelation, rheological properties and in vitro drug release. Formation of in situ gel upon contact with nasal fluid was conferred via the use of ion-activated gellan gum as carrier. In vivo studies in rabbits were performed comparing the nasal bioavailability of CRV after oral, nasal, and intravenous administration. Optimized CRV nanosuspension prepared by combination of poloxamer 407 and oleic acid showed good particle size [d (0.9); 0.19 μm], zeta potential (+10.2 mV) and polydispersity (span; 0.63). The formulation containing 0.5% w/v gellan gum demonstrated good gelation ability and desired sustained drug release over period of 12 h. In vivo pharmacokinetic study revealed that the absolute bioavailability of in situ nasal spray formulation (69.38%) was significantly increased as compared to orally administered CRV (25.96%) with mean residence time 8.65 h. Hence, such in situ gel system containing drug nanosuspension is a promising approach for the intranasal delivery in order to increase nasal mucosal permeability and in vivo residence time which altogether improves drug bioavailability.KEY WORDS: bioavailability, Carvedilol, in situ gel, intranasal, nanosuspension     

Antimicrobial Efficacy of Two Surface Barrier Discharges with Air Plasma against In Vitro Biofilms

The treatment of infected wounds is one possible therapeutic aspect of plasma medicine. Chronic wounds are often associated with microbial biofilms which limit the efficacy of antiseptics. The present study investigates two different surface barrier discharges with air plasma to compare their efficacy against microbial biofilms with chlorhexidine digluconate solution (CHX) as representative of an important antibiofilm antiseptic. Pseudomonas aeruginosa SG81 and Staphylococcus epidermidis RP62A were cultivated on polycarbonate discs. The biofilms were treated for 30, 60, 150, 300 or 600 s with plasma or for 600 s with 0.1% CHX, respectively. After treatment, biofilms were dispensed by ultrasound and the antimicrobial effects were determined as difference in the number of the colony forming units by microbial culture. A high antimicrobial efficacy on biofilms of both plasma sources in comparison to CHX treatment was shown. The efficacy differs between the used strains and plasma sources. For illustration, the biofilms were examined under a scanning electron microscope before and after treatment. Additionally, cytotoxicity was determined by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay with L929 mouse fibroblast cell line. The cell toxicity of the used plasma limits its applicability on human tissue to maximally 150 s. The emitted UV irradiance was measured to estimate whether UV could limit the application on human tissue at the given parameters. It was found that the UV emission is negligibly low. In conclusion, the results support the assumption that air plasma could be an option for therapy of chronic wounds.     

Cell Surface Concentrations and Concentration Ranges for Testing In Vitro Autocrine Loops and Small Molecules

A common assumption made when performing in vitro cellular assays is that the concentration of substances in the culture system is uniform. However, since the cells that internalize and secrete substances reside at the bottom of the well, it is conceivable that a concentration gradient could arise across the fluid layer. Importantly, the concentration of a substance in the vicinity of a cell, which is the concentration of interest, cannot be measured via existing methods. In this work a simple strategy for estimating the concentration of a chemical species at the surface of a cell is presented. Finally, this result is used to outline a method for determining the appropriate concentration ranges for testing in vitro autocrine loops and small molecules.