Membrane proteomes of Pseudomonas aeruginosa and Acinetobacter baumannii.

Acinetobacter baumannii and Pseudomonas aeruginosa are known for their intrinsic resistance to antibiotics. Between mechanisms involved in this resistance, diminished expression of outer membrane proteins and up-regulation of efflux pumps play an important role. The characterization of membrane proteins is consequently necessary because of their importance in the antibiotic resistance but also in virulence. This review presents proteomic investigations aiming to describe the protein content of the membranes of these two bacterial species.

Outer-membrane proteomic maps and surface-exposed proteins of Legionella pneumophila using cellular fractionation and fluorescent labelling.

Bacterial surface-associated proteins play crucial roles in host-pathogen interactions and pathogenesis. The identification of these proteins represents an important goal of bacterial proteomics for vaccine development, but also for environmental concerns such as microbial biosensing. Here, we developed such an approach for Legionella pneumophila, a bacterium that causes severe pneumonia. We propose a complementary strategy consisting of (1) a fluorescent labelling of surface-exposed proteins in parallel with (2) a fractionation of the outer-membrane protein extract. These two distinct protein populations were subsequently separated using two-dimensional gel electrophoresis and characterised by mass spectrometry. Within these populations, we found proteins which were expected for the compartments studied, but also a great number of proteins never experimentally described, and also a non-negligible fraction of proteins never described in these fractions. These data provided new routes of inspection for transport and host recognition for Legionella pneumophila. In addition, these results on the membranome and surfaceome show that Legionella in the stationary phase of growth possesses the major determinants to infect host cells.

Recurrent recovery of Pseudomonas oryzihabitans strains in a karstified chalk aquifer.

Pseudomonas oryzihabitans is an uncommon pathogen that may cause catheter-associated infections, particularly in immunocompromised patients. Although it has been isolated from environment, the source of human infection is not well documented. In the present study, 14 isolates of P. oryzihabitans were recovered over a 28-month period from a karstified chalk aquifer, allowing to advance that distributed natural water could be a source of contamination. Microbiological analyses showed that the bacterium was mainly associated with suspended particulate matters. To investigate the clonality of P. oryzihabitans environmental isolates, 16S rRNA gene sequencing, antibiogram and randomly amplified polymorphic DNA (RAPD) typings were performed. Results demonstrated (i) the presence of at least three clones within the aquifer and (ii) that the presence of the bacterium in groundwater is not only the result of a biofilm bloom but also of an exogenous contamination.

Long-term incomplete xylose fermentation, after glucose exhaustion, with Candida shehatae co-immobilized with Saccharomyces cerevisiae.

Saccharomyces cerevisiae and Candida shehatae were co-immobilized in an agar sheet which was introduced in an original two-chambered bioreactor asymmetrically fed in a batch mode with a mixture of glucose and xylose in a ratio of 35:15. The two sugars were consumed simultaneously. All glucose was fermented but only 20% of xylose. After incubation, yeast cells recovered from different areas of the agar sheet (close to, called Hi, and distant from, called Ho, the substrate chamber) were cultured as suspended cells in fresh culture medium provided with xylose or the sugar mixture. Xylose utilization by gel released Hi yeasts was significantly delayed compared to the Ho culture. Ethanol consumption by Hi yeasts in the two-substrate medium occurred after glucose exhaustion despite the presence of xylose. The waste medium resulting from incubation of the immobilized-cell structure inhibited xylose utilization by C. shehatae. Our results suggested that batch fermentation most probably favoured this incomplete xylose fermentation.

Proteomic analysis of residual proteins in blades and petioles of fallen leaves of Brassica napus.

Brassica napus L. is an important crop plant, characterised by high nitrogen (N) levels in fallen leaves, leading to a significant restitution of this element to the soil, with important consequences at the economic and environmental levels. It is now well established that the N in fallen leaves is due to weak N remobilisation that is especially related to incomplete degradation of foliar proteins during leaf senescence. Identification of residual proteins in a fallen leaf (i.e. incompletely degraded in the last step of the N remobilisation process) constitutes important information for improving nutrient use efficiency. Proteome analysis of the vascular system (petioles) and blades from fallen leaves of Brassica napus was performed, and the 30 most abundant residual proteins in each tissue were identified. Among them, several proteins involved in N recycling remain in the leaf after abscission. Moreover, this study reveals that some residual proteins are associated with energy metabolism, protection against oxidative stress, and more surprisingly, photosynthesis. Finally, comparison of blade and petiole proteomes show that, despite their different physiological roles in the non-senescing leaf, both organs redirect their metabolism in order to ensure catabolic reactions. Taken together, the results suggest that a better degradation of these leaf proteins during the senescence process could enable improvements in the N use efficiency of Brassica napus.

Do beta-lactam antibiotics permeabilize the outer membrane of gram-negative bacteria? An electrochemical investigation.

The effects of cefotaxime and EDTA on the reducing activity of Escherichia coli and Staphylococcus aureus cultures growing in the presence of lipoic acid (LA) were investigated by potential-time measurements. The potentiometric responses of E. coli cultures exposed to EDTA indicated enhanced transmembrane transport of LA as a consequence of the outer membrane permeabilization by the chelator, whereas EDTA exerted no effect on the reducing activity of S. aureus cultures. In the same way, cefotaxime stimulated the reducing activity of E. coli, but not that of S. aureus. These results suggest that cefotaxime, and, more generally, a great variety of beta-lactam antibiotics, are able to permeabilize the outer membrane of Gram-negative bacteria.

Agar-entrapped bacteria as an in vitro model of biofilms and their susceptibility to antibiotics.

A simple in vitro system was developed as a model structure of biofilms and to evaluate their susceptibility to antibiotics. Viable Escherichia coli cells were entrapped in agar gel layers and incubated for 2 days in a minimal salt medium supplemented with glucose. After subsequent culture for 3 weeks under metal ion depletion, the biomass distribution inside the gel layer was highly heterogeneous. The cell concentration reached 10(11) cfu/g gel in the outer regions of the agar structure whereas the inner gel areas were less colonized (10(9) cfu/g gel). Immobilized cells displayed enhanced resistance to latamoxef as compared with free microorganisms. Moreover, a 3-week-old immobilized-cell membrane was less susceptible to the antibiotic than a younger (2 days old) one. The exposure for 11 h to 64 micrograms/cm3 latamoxef killed about 90% of the bacteria entrapped in the older agar layer, whereas the number of killed cells was 100-fold higher in the younger structure. Effective diffusivity measurements showed that the diffusion of latamoxef in the biofilm-like agar structures was moderately restricted as compared to that in water, and independent of the immobilized-cell content.

Occurrence of sessile Pseudomonas oryzihabitans from a karstified chalk aquifer.

Pseudomonas oryzihabitans is an uncommon pathogen that may cause opportunistic infections. Although it has been previously isolated from the environment, the source of human infection has not been well documented. In this study, we describe the presence of P. oryzihabitans adhering on suspended particulate matters recovered from karst groundwaters. The isolated pathogen was capable of forming biofilms on silicon supports and clay beads. Adherent P. oryzihabitans cells displayed a high resistance to chlorine as compared with the same organisms cultured in the planktonic mode. These results demonstrate that aquifer biofilms are potential environmental sources for water-born P. oryzihabitans infections and that bacterial attachment might affect drinking water purification.

Susceptibility of agar-entrapped Escherichia coli cultures to cefotaxime as assessed by the potentiometric measurement of lipoic acid reduction.

We used an original potentiometric procedure to monitor the metabolic activity of biofilm-like structures and to assess their susceptibility to betalactam antibiotics. The reduction of exogeneous alpha-lipoic acid to dihydrolipoic acid by planktonic and agar-entrapped bacteria, metabolizing glucose, was followed by potential-time measurements using gold versus reference electrodes. Immobilized cultures displayed a lower susceptibility to cefotaxime than their free counterparts. The redox potential-time courses showed that the gel matrix was more rapidly oxygen depleted than the free-cell culture medium. The depletion of both glucose and dissolved O2 inside the gel induced a breakdown in the reducing activity of agar-entrapped organisms.

Towards a better understanding of biomarker response in field survey: a case study in eight populations of zebra mussels.

In order to provide reliable information about responsiveness of biomarkers during environmental monitoring, there is a need to improve the understanding of inter-population differences. The present study focused on eight populations of zebra mussels and aimed to describe how variable are biomarkers in different sampling locations. Biomarkers were investigated and summarised through the Integrated Biomarker Response (IBR index). Inter-site differences in IBR index were analysed through comparisons with morphological data, proteomic profiles and genetic background of the studied populations. We found that the IBR index was a good tool to inform about the status of sites. It revealed higher stress in more polluted sites than in cleaner ones. It was neither correlated to proteomic profiles nor to genetic background, suggesting a stronger influence of environment than genes. Meanwhile, morphological traits were related to both environment and genetic background influence. Together these results attest the benefit of using biological tools to better illustrate the status of a population and highlight the need of consider inter-population difference in their baselines.

Quantitative subproteomic analysis of germinating related changes in the scutellum oil bodies of Zea mays.

Oil bodies (OBs) were purified from the scutellum of mature maize embryos and from embryos 2 days after imbibition and their associated proteins were extracted and separated by 2-DE. Eighteen proteins were shown to be differentially accumulated, thirteen showed a higher accumulation in mature scutellum and five were highly accumulated in the germinating scutellum. Proteins were identified using LC-MS/MS. Besides previously known oil body protein oleosin, other proteins were identified in this study. Among accumulated proteins during imbibition are prohibitin 2, stress-inducible membrane pore protein Tim17 and manganese superoxide dismutase. Among the proteins whose amount decreases during imbibition are cupin 2, two different protein disulfide isomerases, a triosephosphate isomerase, a class IV heat shock protein, the embryonic protein DC-8, the 60S ribosomal protein P0, a nucleoside-diphosphate kinase, and a rubber elongation factor protein. Some of the identified proteins were previously located in organelles other than oil bodies, suggesting that OBs may interact with these organelles. We also suggest that OBs may act as transient storage depots for proteins that are temporally in excess.

Antibacterial surfaces developed from bio-inspired approaches.

Prevention of bacterial adhesion and biofilm formation on the surfaces of materials is a topic of major medical and societal importance. Various synthetic approaches based on immobilization or release of bactericidal substances such as metal derivatives, polyammonium salts and antibiotics were extensively explored to produce antibacterial coatings. Although providing encouraging results, these approaches suffer from the use of active agents which may be associated with side-effects such as cytotoxicity, hypersensibility, inflammatory responses or the progressive alarming phenomenon of antibiotic resistance. In addition to these synthetic approaches, living organisms, e.g. animals and plants, have developed fascinating strategies over millions of years to prevent efficiently the colonization of their surfaces by pathogens. These strategies have been recently mimicked to create a new generation of bio-inspired biofilm-resistant surfaces. In this review, we discuss some of these bio-inspired methods devoted to the development of antibiofilm surfaces. We describe the elaboration of antibacterial coatings based on natural bactericidal substances produced by living organisms such as antimicrobial peptides, bacteriolytic enzymes and essential oils. We discuss also the development of layers mimicking algae surfaces and based on anti-quorum-sensing molecules which affect cell-to-cell communication. Finally, we report on very recent strategies directly inspired from marine animal life and based on surface microstructuring.

Electrochemical monitoring of chlorhexidine digluconate effect on polyelectrolyte immobilized bacteria and kinetic cell adhesion.

The electrochemical impedance spectroscopy (EIS) technique has been used as a sensitive method to explore the effect of antibacterial molecules on immobilized bacteria and biofilm formation. In this work, we describe the electrochemical spectroscopy as a powerful method to monitor the effect of chlorhexidine digluconate (CHX-Dg) on polyelectrolyte immobilized Escherichia coli K12 MG1655 and the kinetics of cell adhesion on gold electrodes. The experimental impedance data were modeled with a Zview program to find the best equivalent electrical circuit and analyse its parameter's properties. Polyelectrolyte multilayer formation on the electrode surface and bacteria immobilization greatly increased the electron-transfer resistance (R(et)) and reduced the constant phase element (CPE(dl)). The effect of CHX-Dg was studied in a 0.5 x 10⁻4 mmol l⁻¹ to 0.5 mmol l⁻¹ range. The relation between the evolution of R(et) and CHX-Dg concentration was found to be negatively correlated. When CHX-Dg was added, the electrochemical monitoring of the bacterial kinetic adhesion showed that the electrode's capacity (C(P)) variation remained stable, demonstrating that the addition of CHX-Dg in the broth inhibited bacterial adhesion.

Selective detection and enumeration of fecal coliforms in water by potentiometric measurement of lipoic acid reduction.

Water samples of various origins were inoculated into a specific coliform-selective lactose broth provided with lipoic (thioctic) acid, and the time evolution of the redox potential of the cultures was monitored during incubation at 41 degrees C by use of gold versus reference electrodes. Positive potential-time responses, i.e., 100-mV potential shifts recorded within 20 h of inoculation, were related to the initial number of fecal coliforms in the broth determined by control enumeration techniques, and the organisms responsible were isolated and identified by conventional procedures. A total of 30 samples of wastewater, 38 of surface water, 553 of groundwater, and 110 of drinking water were tested successively. A total of 240 natural water samples, including 172 groundwater samples, and 1 drinking water sample were found to be positive in the potentiometric test. The majority (i.e., 92.5%) of the relevant potentiometric detection times were shorter than 15 h, and 96% of these could be attributed to Escherichia coli. Fifteen hours corresponded to the limit for detecting 1 E. coli cell per 100 ml of water. About 78% of the potentiometric responses occurring after 15 h were induced by fecal coliforms other than E. coli (Enterobacter cloacae, Klebsiella pneumoniae, and Citrobacter freundii). Calibration curves relating detection times shorter than 15 h to fecal coliform (i.e., E. coli) concentrations were constructed for the natural water samples tested. There were minor variations in the average growth rate of the organisms in the relation to the contamination level of the water tested. The number of false-positive samples in the potentiometric test was equivalent to that of false-negative samples (groundwater or drinking water).

Cell immobilization in composite agar layer microporous membrane structures: growth kinetics of gel-entrapped cultures and cell leakage limitation by a microporous membrane.

Agar discs containing different amounts of viable Escherichia coli cells (from 10 to 10(6) organisms.g-1 agar) were incubated in a nutrient medium and the growth of agar-entrapped bacteria and free (released) cells was monitored. The study was repeated with composite immobilized-cell structures obtained by placing a microporous membrane filter between the gel matrix and the incubation medium. In both cases, immobilized cells grew exponentially and reached a peak concentration an order of magnitude higher than that of free (suspended) cell cultures. The maximum specific growth rates of entrapped bacteria, ranging between 0.0115 min-1 and 0.0145 min-1, i.e., slightly higher than that of control free cultures (0.011 min-1), showed no clear dependence on the initial cell loading (ICL). The microporous filter proved efficient in limiting cell leakage since it noticeably lengthened the leakage time at a given ICL. This efficiency, however, decreased at high ICL and high growth rate of immobilized organisms.

Antibacterial efficacy of tobramycin against anaerobic Escherichia coli cultures in the presence of electron acceptors.

The antimicrobial activity of tobramycin against anaerobic cultures of Escherichia coli was tested in the presence of various electron carriers. The presence of 2,6-dichlorophenol 4-indophenol (DCIP) significantly enhanced the killing efficacy of tobramycin. Only 0.003% of the initial cell population (i.e. 10(6) cfu/mL) remained viable after exposure for 10 h to the mixture of antibiotic (20 x MIC, i.e. 40 mg/L) and electron acceptor (10(-3) M), as compared with 9% of surviving organisms in the presence of tobramycin alone. Less synergy was obtained with p-benzoquinone and 1,2-naphthoquinone. Fumarate did not affect the efficiency of the antibiotic. The mixture of tobramycin and DCIP was ineffective against agar-entrapped bacteria which, like biofilm organisms, are subject to oxygen limitation.

Underexpression of porin protein OmpF in agar-entrapped, sessile-like Escherichia coli.

The electrophoretic patterns of the outer membrane proteins of agar-entrapped Escherichia coli cells were found to be different from those of free organisms. In particular, the porin protein OmpF was underexpressed in immobilized bacteria, that displayed enhanced resistance to latamoxef.

Protein synthesis in Escherichia coli at 4 degrees C.

Two-dimensional electrophoresis technology was used to investigate protein synthesis by the mesophilic bacterium Escherichia coli at low temperature. It was confirmed that protein synthesis in E. coli decreased strongly after a temperature downshift from 37 to 4 degrees C. After incubation for 150 min at 4 degrees C, however, the number of synthesized proteins represented 60% of the overall polypeptide number observed at 37 degrees C. Furthermore, the analysis of autoradiograms revealed the overexpression of 69 proteins by shocked bacteria, showing that the number of cold-induced proteins has been significantly underestimated so far.

The role of oxygen limitation in the resistance of agar-entrapped, sessile-like Escherichia coli to aminoglycoside and beta-lactam antibiotics.

Viable cells of Escherichia coli were entrapped in agar gel layers to form artificial biofilm-like structures. Killing assays of immobilized organisms by latamoxef and tobramycin were performed under different oxygenation conditions of the culture medium and compared with free-cell experiments. Under moderate aeration, agar-entrapped bacteria displayed higher resistance to the two antibiotics than suspended cells. Slow-growing free-cell cultures were resistant to latamoxef but not to tobramycin. In anaerobic incubation conditions, suspended organisms were highly resistant to the two antibiotics. Sustained oxygenation enhanced tobramycin efficacy against free and immobilized cells. These results show that oxygen deficiency in the gel layer contributes to the enhanced antibiotic resistance of sessile-like cells.

Continuous alcoholic fermentation of glucose/xylose mixtures by co-immobilized Saccharomyces cerevisiae and Candida shehatae.

Viable Saccharomyces cerevisiae and Candida shehatae cells were co-immobilized in a composite agar layer/microporous membrane structure. This immobilized-cell structure was placed in a vertical position between the two halves of a double-chambered, stainless-steel bioreactor of original design and applied to the continuous alcoholic fermentation of a mixture of glucose (35 g dm-3) and xylose (15 g dm-3). Various dilution rates and initial cell loadings of the gel layer were tested. Simultaneous consumption of the two sugars was always observed. The best fermentation performance was obtained at low dilution rate (0.02 h-1) with an excess of C. shehatae over S. cerevisiae in the initial cell loading of the gel (5.0 mg dry weight and 0.65 mg dry weight cm-3 gel respectively): 100% of glucose and 73% of xylose were consumed with an ethanol yield coefficient of 0.48 g g total sugars-1. In these conditions, however, the ethanol production rate per unit volume of gel remained low (0.37 g h-1 dm-3). Viable cell counts in gel samples after incubation highlighted significant heterogeneities in the spatial distribution of the two yeast species in both the vertical and the transverse directions. In particular, the overall cell number decreased from the bottom to the top of the agar sheet, which may explain the low ethanol productivity relative to the total gel volume.