The attachment of Enteromorpha zoospores to a bacterial biofilm assemblage

This study has investigated the relationship between bacterial biofilms and the attachment of zoospores of the green macroalga Enteromorpha. Zoospore attachment to glass slides was enhanced in the presence of a bacterial biofilm assemblage, and the number attaching increased with the number of bacteria present. Zoospores also attached to control surfaces, but at lower numbers; glass surfaces conditioned in autoclaved seawater had the same number of zoospores attached as new glass surfaces. The spatial relationship between bacterial cells and attached zoospores was quantified by image analysis. The hypothesis tested was that zoospores attached preferentially to, or in the very close vicinity of, bacterial cells. Spatial microscopic analysis showed that more bacteria were covered by zoospores than would be expected if zoospore attachment was a random process and zoospores appeared to attach to bacterial clusters. The most likely explanation is that zoospores are attracted to bacterial cells growing on surfaces and the presence of a bacterial biofilm enhances their settlement. The possibility is discussed that Enteromorpha zoospores respond to a chemical signal produced by bacteria, i.e. that there may be prokaryote‐eukaryote cell signalling.     

Role of bacterial cellulose fibrils in Agrobacterium tumefaciens infection.

During the attachment of Agrobacterium tumefaciens to carrot tissue culture cells, the bacteria synthesize cellulose fibrils. We examined the role of these cellulose fibrils in the attachment process by determining the properties of bacterial mutants unable to synthesize cellulose. Such cellulose-minus bacteria attached to the carrot cell surface, but, in contrast to the parent strain, with which larger clusters of bacteria were seen on the plant cell, cellulose-minus mutant bacteria were attached individually to the plant cell surface. The wild-type bacteria became surrounded by fibrils within 2 h after attachment. No fibrils were seen with the cellulose-minus mutants. Prolonged incubation of wild-type A. tumefaciens with carrot cells resulted in the formation of large aggregates of bacteria, bacterial fibrils, and carrot cells. No such aggregates were formed after the incubation of carrot cells with cellulose-minus A. tumefaciens. The absence of cellulose fibrils also caused an alteration in the kinetics of bacterial attachment to carrot cells. Cellulose synthesis was not required for bacterial virulence; the cellulose-minus mutants were all virulent. However, the ability of the parent bacterial strain to produce tumors was unaffected by washing the inoculation site with water, whereas the ability of the cellulose-minus mutants to form tumors was much reduced by washing the inoculation site with water. Thus, a major role of the cellulose fibrils synthesized by A. tumefaciens appears to be anchoring the bacteria to the host cells, thereby aiding the production of tumors.     

Rapid,in situ detection of Agrobacterium tumefaciens attachment to leaf tissue

Attachment of the plant pathogen Agrobacterium tumefaciens to host plant cells is an early and necessary step in plant transformation and agroinfiltration processes. However, bacterial attachment behavior is not well understood in complex plant tissues. Here we developed an imaging‐based method to observe and quantify A. tumefaciens attached to leaf tissue in situ. Fluorescent labeling of bacteria with nucleic acid, protein, and vital dyes was investigated as a rapid alternative to generating recombinant strains expressing fluorescent proteins. Syto 16 green fluorescent nucleic acid stain was found to yield the greatest signal intensity in stained bacteria without affecting viability or infectivity. Stained bacteria retained the stain and were detectable over 72 h. To demonstrate in situ detection of attached bacteria, confocal fluorescent microscopy was used to image A. tumefaciens in sections of lettuce leaf tissue following vacuum‐infiltration with labeled bacteria. Bacterial signals were associated with plant cell surfaces, suggesting detection of bacteria attached to plant cells. Bacterial attachment to specific leaf tissues was in agreement with known leaf tissue competencies for transformation with Agrobacterium. Levels of bacteria attached to leaf cells were quantified over time post‐infiltration. Signals from stained bacteria were stable over the first 24 h following infiltration but decreased in intensity as bacteria multiplied in planta. Nucleic acid staining of A. tumefaciens followed by confocal microscopy of infected leaf tissue offers a rapid, in situ method for evaluating attachment of A. tumefaciens' to plant expression hosts and a tool to facilitate management of transient expression processes via agroinfiltration. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step - bacterial growth - is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane (PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.     

Automatic enumeration of adherent streptococci or actinomyces on dental alloy by fluorescence image analysis.

The aim of the present study was to develop an automated image analysis method to quantify adherence of Streptococcus sanguinis or Actinomyces viscosus on surfaces of a currently used dental alloy. Counting such bacterial strains was difficult because of their arrangement, thus S. sanguinis being a coccus arranged in chains or pairs, and A. viscosus a long complexly arranged polymorph rod. Direct counting of fluorescently stained adherent bacteria was done visually and with image analysis methods. To differentiate these two morphotypes, two programs were developed: (i) for streptococci, thresholding and selection of the object maxima, and (ii) for actinomyces, two step thresholding and processing of the characteristic points of the object skeletons. The triplicate enumerations for each bacterial strain were not significantly different (p > 0.005) and correlations between visual counting and automated counting were significant (r = 0.91 for S. sanguinis and r = 0.99 for A. viscosus, p <00.0001). These rapid and reproducible methods, allowed us to count either cocci or rods, adherent on an inert substratum, in high density conditions.     

Binary and mixed population biofilms: Time-lapse image analysis and disinfection with biocides

Simultaneous binary population biofilm formation by a bacterium and filamentous fungus was demonstrated by time-lapse image analysis in a flow cell system. The accumulation of attached bacterial cells followed an S-shaped graph similar to batch culture bacterial growth, with continual attachment, detachment, rotation, and movement of bacteria over the surface. An extensive hyphal network formed on the surface of the flow cell, protruding into the bulk flow, which subsequently detached. Multiple species mixed fungal–bacterial model biofilms were tested for isothiazolone biocide susceptibility. Biofilms were less susceptible to biocide treatment than planktonic cells of the same organisms. Mixed species biofilms, particularly for the bacterial species, offered greater protection against the action of the biocide compared to single species biofilms. Microbial loss as a result of biocide activity was shown by reduced cell surface coverage in electron micrographs. Received 11 March 2002/ Accepted in revised form 08 August 2002     

IMAGE ANALYSIS AS A RAPID PATHOGEN DETECTION METHOD FOR USE IN POULTRY INDUSTRY

An image analysis system was developed and evaluated as a method for rapid detection of Salmonella typhimurium in pure culture and in chicken washes. A direct immunomagnetic separation and immunofluorescent staining technique was developed to capture and identify target cells. Digital images were acquired and segmented into background and bacteria. Bacteria were enumerated using a custom designed image analysis software. The image analyses results were compared with manual enumeration. A correlation coefficient of 0.78 was established between manual and image analysis counts. In addition, the difference between the manual and the image analysis bacterial counts in individual images was low. Image analysis took an average of 15 s to analyze an image. The results indicate that the proposed system has the potential to be used as a rapid screening procedure for bacterial detection in the food industry.     

Detection, enumeration, and sizing of planktonic bacteria by image-analyzed epifluorescence microscopy.

Epifluorescence microscopy is now being widely used to characterize planktonic procaryote populations. The tedium and subjectivity of visual enumeration and sizing have been largely alleviated by our use of an image analysis system consisting of a modified Artek 810 image analyzer and an Olympus BHT-F epifluorescence microscope. This system digitizes the video image of autofluorescing or fluorochrome-stained cells in a microscope field. The digitized image can then be stored, edited, and analyzed for total count or individual cell size and shape parameters. Results can be printed as raw data, statistical summaries, or histograms. By using a stain concentration of 5 micrograms of 4'6-diamidino-2-phenylindole per ml of sample and the optimal sensitivity level and mode, counts by image analysis of natural bacterial populations from a variety of habitats were found to be statistically equal to standard visual counts. Although the time required to prepare slides, focus, and change fields is the same for visual and image analysis methods, the time and effort required for counting is eliminated since image analysis is instantaneous. The system has been satisfactorily tested at sea. Histograms of cell silhouette areas indicate that rapid and accurate estimates of bacterial biovolume and biomass will be possible with this system.     

Detection, enumeration, and sizing of planktonic bacteria by image-analyzed epifluorescence microscopy

Epifluorescence microscopy is now being widely used to characterize planktonic procaryote populations. The tedium and subjectivity of visual enumeration and sizing have been largely alleviated by our use of an image analysis system consisting of a modified Artek 810 image analyzer and an Olympus BHT-F epifluorescence microscope. This system digitizes the video image of autofluorescing or fluorochrome-stained cells in a microscope field. The digitized image can then be stored, edited, and analyzed for total count or individual cell size and shape parameters. Results can be printed as raw data, statistical summaries, or histograms. By using a stain concentration of 5 micrograms of 4'6-diamidino-2-phenylindole per ml of sample and the optimal sensitivity level and mode, counts by image analysis of natural bacterial populations from a variety of habitats were found to be statistically equal to standard visual counts. Although the time required to prepare slides, focus, and change fields is the same for visual and image analysis methods, the time and effort required for counting is eliminated since image analysis is instantaneous. The system has been satisfactorily tested at sea. Histograms of cell silhouette areas indicate that rapid and accurate estimates of bacterial biovolume and biomass will be possible with this system.     

Combining flow cytometry and gfp reporter gene for quantitative evaluation of Pectpbacterium carotovorum ssp. carotovorum in Ornithogalum dubium plantlets

Aims: Ornithogalum dubium is a natural host of the soft rot pathogen Pectobacterium carotovorum ssp. carotovorum (Pcc). The present study was aimed to develop a quantification system for Pcc expressing a gfp reporter gene, using fluorescent activated cell sorter (FACS) in planta. Methods and Results: Several calibration steps were required to distinctly gate the GFP‐labelled bacteria at FL1 mode and count the bacteria. To validate the bacterial counts obtained by FACS analysis, an internal standard of polystyrene green fluorescent microsphere beads was employed, resulting in high correlation with serial dilutions and plate counting. This allowed quantification of the bacteria, with no further need to culture, dilute or plate the cells. Micropropagation tools were developed to produce uniform plantlets of O. dubium, which were either inoculated with increasing concentrations of Pcc or elicited for resistance towards Pcc using methyl jasmonate. The rapid counting procedure allowed recovering, gating and counting the bacterial population in planta, separately from the plant cells background and from the microsphere beads. Conclusions: The FACS based quantification approach of Pcc was found accurate, reproducible and time saving, thus useful for counting bacteria in planta. Significance and Impact of the Study: The combination of time‐ and cost‐saving approach for Pcc quantification with efficient screening tools during early stages of micropropagation may facilitate the preliminary process of selection for resistant cultivars.     

Elaboration of cellulose fibrils by Agrobacterium tumefaciens during attachment to carrot cells.

The attachment of virulent strains of Agrobacterium tumefaciens to plant cells is the first step in the bacterial induction of tumors. Binding of A. tumefaciens to carrot tissue culture cells occurred as a two-step process. The initial step was the attachment of the bacteria to the plant cell wall. Living plant cells were not required. Bacterial attachment to heat-killed or glutaraldehyde-fixed carrot cells proceeded with only slightly altered kinetics and unaltered bacterial strain specificity. After the bacteria bound to the carrot cell surface, scanning electron microscopy showed that fibrils developed, surrounded the bacteria, and anchored them to the plant cell surface. These fibrils were synthesized by the bacteria and not by the plant cell since they were also made after the attachment of A. tumefaciens to dead carrot cells and since under some conditions the bacteria synthesized fibrils in the absence of plant cells. Calcofluor staining, acid hydrolysis, enzymatic digestion studies, and infrared spectroscopy showed that the fibrils were composed of cellulose. The formation of these cellulose fibrils occurred during the attachment of virulent strains of A. tumefaciens to plant cells in vitro. The fibrils anchored the bacteria to the plant cell surface and entrapped additional bacteria. The multiplication of entrapped and attached bacteria resulted in the formation of large clusters of bacteria held close to the plant cell wall and plasma membrane by cellulose fibrils. This high concentration of bacteria may facilitate transfer of Ti plasmid deoxyribonucleic acid to the plant cell resulting in the formation of tumors.     

Rumen bacterial interrelationships with plant tissue during degradation revealed by transmission electron microscopy

The mode of rumen bacterial degradation of cell walls in coastal bermudagrass [Cynodon dactylon (L) Pers.] differed with the plant tissue type. Bacteria degraded thin, primary cell walls of mesophyll and phloem apparently by extracellular enzymes and without prior attachment; thick-walled bundle sheath and epidermal cells apparently were degraded after bacterial attachment, in some types by an extracellular substance, to the plant cell walls. Rumen bacteria split the nondegraded cuticle from the epidermis by preferentially attacking the cell just underneath the cuticle. The propensity for bacterial attachment to lignified cells of the vascular tissue was low, and bacterial degradation of these cells did not occur after 72 h of incubation.     

An improved method for the automated enumeration of fluorescently labelled bacteria in human faeces

The study aimed to improve microscopy-based automated recognition of faecal bacterial cells labelled with 16S rRNA-targeted oligonucleotides and 4',6-diamidino-2-phenylindole (DAPI). Based on the software KS400 (Carl Zeiss Vision, Hallbergmoos, Germany), designed for automising microscopy-based image capture and image analysis, a routine was developed that affords the recognition of doubly stained bacteria and the rejection of artefacts. The accuracy of the automated enumeration was investigated by comparing the resulting data with those obtained by manual counting. The newly developed method was subsequently used to compare the total bacterial counts in human faecal samples using the domain specific probe Eub338 alone and a mixture of 5 domain-specific probes, respectively. Faecal samples from 90 healthy volunteers were analysed. The cell counts obtained with Eub338 were 10% lower than those obtained with the probe mixture. Since the cells detected with the probe mixture covered a wide range of signal intensities, a dynamic analysis routine was developed to effectively detect the whole range of bright to weak signals within the same image, while at the same time reliably rejecting artefacts.     

Fabrication of titanium based MALDI bacterial chips for rapid, sensitive and direct analysis of pathogenic bacteria

For the first time, we report the fabrication of a titanium bacterial chip for MALDI-MS produced from a simple, cost effective and rapid heat treatment process. This bacterial chip can be reused many times and is highly versatile. These bacterial chips serve dual roles: (1) They can be applied as MALDI-MS target plates for direct and highly sensitive bacterial analysis. (2) They can be used as bacterial sensors for direct analysis of the captured bacteria using MALDI-MS. The sensitivity of these chips when used as bacterial sensors is <10(3)cfu/mL. The lowest detectable concentration for direct MALDI-MS analysis was found to be 10(4)cfu/mL. The results were further justified by using standard plate counting method combined with Tukey-Kramer statistical analysis and fluorescence imaging followed by image processing for fluorescence quantification using ImageJ software to substantiate the MALDI-MS results.     

Direct in situ viability assessment of bacteria in probiotic dairy products using viability staining in conjunction with confocal scanning laser microscopy

The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD BacLight staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD BacLight staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was approximately 10(8) bacteria/ml (equivalent to approximately 10(7) CFU/ml), based on Bifidobacterium sp. strain UCC 35612 counts in maximum-recovery diluent.     

Interaction of Pseudomonas solanacearum with Suspension-Cultured Tobacco Cells and Tobacco Leaf Cell Walls In Vitro

Attachment of radiolabeled Pseudomonas solanacearum cells to suspension-cultured tobacco cells and tobacco leaf cell walls was measured in vitro by a filtration technique that allowed separation of attached and unattached bacteria. An avirulent strain (B1) attached more rapidly to suspension-cultured cells than did the virulent parent strain (K60), and B1 attachment was less sensitive to inhibition by high ionic strength than was K60. Attachment of B1 bacteria to suspension-cultured cells and to leaf cell walls was comparable (50 to 70%), but only a small proportion (10 to 20%) of K60 bacteria attached to leaf cell walls under optimal conditions. With high bacterial populations (10⁸ bacteria per ml), attachment of K60 to suspension-cultured cells was greatly reduced. Attachment of both strains was completely inhibited by pretreating bacterial cells with heat (41°C) or azide and was partially inhibited by EDTA and kanamycin. The mechanism of attachment is not known, but ionic forces may be involved.     

Monitoring of microbial adhesion and biofilm growth using electrochemical impedancemetry

Electrochemical impedance spectroscopy was tested to monitor the cell attachment and the biofilm proliferation in order to identify characteristic events induced on the metal surface by Gram-negative (Pseudomonas aeruginosa PAO1) and Gram-positive (Bacillus subtilis) bacteria strains. Electrochemical impedance spectra of AISI 304 electrodes during cell attachment and initial biofilm growth for both strains were obtained. It can be observed that the resistance increases gradually with the culture time and decreases with the biofilm detachment. So, the applicability of electric cell-substrate impedance sensing (ECIS) for studying the attachment and spreading of cells on a metal surface has been demonstrated. The biofilm formation was also characterized by the use of scanning electron microscopy and confocal laser scanning microscopy and COMSTAT image analysis. The electrochemical results roughly agree with the microscope image observations. The ECIS technique used in this study was used for continuous real-time monitoring of the initial bacterial adhesion and the biofilm growth. It provides a simple and non-expensive electrochemical method for in vitro assessment of the presence of biofilms on metal surfaces.     

Adherence of Agrobacterium tumefaciens to the cells of immature wheat embryos

Agrobacterium attached to wheat embryos in vitro. This attachment was plasmid independent, and occurred on both wounded and unwounded cell surfaces. The pattern of attachment clearly demonstrated that bacterial attachment to cereal cells follows the same trends observed for dicotyledonous plants. During the inoculation period the bacterial cells attach to the plant cell walls either with lateral or polar orientation. Wounding (mechanical or enzymatic) preferentially promoted adherence of the bacteria at the wound site, however, attachment was not wound dependent.     

Real‐time PCR method for the quantification of Burkholderia cepacia complex attached to lung epithelial cells and inhibition of that attachment

Aims: To develop a rapid method to quantify the attachment of the cystic fibrosis pathogen, Burkholderia multivorans, to lung epithelial cells (16HBE14o^?) using real‐time PCR with a view to monitoring potential inhibition of lung cell attachment. Methods and Results: Mammalian and bacterial DNA were purified from bacteria attached to lung epithelial cells. The relative amount of bacteria attached was determined by amplification of the recA gene relative to the human GAPDH gene, in the presence of SYBR Green^®. The method was thoroughly validated and shown to correlate well with traditional plating techniques. Inhibition of bacterial attachment with simple sugars was then evaluated by real‐time PCR. Of the sugars examined, pre‐incubation of B. multivorans with lactose, mannose and xylitol all decreased bacterial adherence to 16HBE14o^? cells, while glucose and galactose had no significant effect. Pre‐incubation with lactose had the greatest effect, resulting in reduced adhesion to 35% of untreated controls. Conclusions: This method can be used to quickly and effectively screen novel agents with higher affinities for bacterial adhesins. Significance and Impact of the Study: This method will enable the rapid development of novel agents to inhibit colonization by this pathogen from the environment.     

Identification of MEK- and phosphoinositide 3-kinase-dependent signalling as essential events during Chlamydia pneumoniae invasion of HEp2 cells

The ability of Chlamydia pneumoniae to survive and cause disease is predicated on efficient invasion of cellular hosts. While it is recognized that chlamydial determinants are important for mediating attachment and uptake into non-phagocytic cells, little is known about the bacterial ligands and cellular receptors that facilitate invasion or host cell signal transduction pathways implicated in this process. We used transmission and scanning electron microscopy to demonstrate that attachment of bacteria to host cells induced the appearance of microvilli on host cell membranes. Invasion occurred 30-120 min after cell contact with the subsequent loss of membrane microvilli. Using an epithelial cell infection model, C. pneumoniae invasion caused a rapid and sustained increase in MEK-dependent phosphorylation and activation of ERK1/2, followed by PI 3-kinase-dependent phosphorylation and activation of Akt. Tyrosine phosphorylation of focal adhesion kinase (FAK) preceded its appearance in a complex with the p85 subunit of PI 3-kinase during chlamydial invasion and isoform-specific tyrosine phosphorylation of the docking protein Shc also occurred at the time of attachment and entry of bacteria. Chlamydia entry but not attachment could be abrogated with specific inhibitors of MEK, PI 3-kinase and actin polymerization, demonstrating the importance of these signalling pathways and an intact actin cytoskeleton for C. pneumoniae invasion. These results suggest that activation of specific cell signalling pathways is an essential strategy used by C. pneumoniae to invade epithelial cells.